(Received for publication, June 1, 1984). Roger A. Johnson$& Karl H. Jakobsll, and Gunter Schultzll. From the $Department of Physiology, Vanderbilt University ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY (0 1985 by The American Society of Biological Chemists, Inc.
Vol. 260, No. 1, Issue of January 10, pp. 114-121,1385 Printed in U.S.A.
Extraction of the Adenylate Cyclase-activating Factorof Bovine Sperm andIts Identification as a Trypsin-like Protease* (Received for publication, June 1, 1984)
Roger A. Johnson$& Karl H. Jakobsll, and GunterSchultzll From the $Department of Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee37232 and the YPharmakologisches Imtitut der Universitat Heidelberg, Im Neuenheimer Feld 366, 6900 Heidelberg, Federal Republic of Germany
We previously reported the activation of adenylate ing maximal stimulation being: 200 ng trypsin/ml; 2 cyclases from rat brain (Johnson, R. A,, Awad, J. A., pg a-chymotrypsin/ml; and 2 pg acrosin/ml. By com(7pg protein/ Jakobs, K. H., and Schultz,G., (1983)FEBSLett. 152, parison, the crude extracted sperm factor 11-16)and from human platelets (Jakobs,K. H., John- ml) enhanced guanosine 5’-0-(thiotriphosphate)-stimson, R. A., and Schultz, G. (1983)Biochim. Biophys. ulated cyclase activity approximately 26-fold. The Acta 756, 369-375) by a factor derived from bovine data suggest that thesperm protease described here is sperm. In this report we describe the conditions for the more effective than acrosin and may be distinct from extraction of the factor frombovine sperm and char- it. acteristics of its effects on adenylate cyclase which are consistent with its being a protease. The activating capacity of sperm particles was extracted from previously washed and frozen sperm into a 30,000 x g The adenylate cyclase (ATP:pyrophosphate lyase, cyclizing; supernatant fraction by various salts, but not by the EC 4.6.1.1) of mammalian sperm is a membrane-bound ennonionic detergent Lubrol-PX. The amount of ex- zyme which exhibits markedly greater activity with Mn2+ tracted factor: (a)was greatest withNH,HCO, > NaCl than with Mg2+ and is insensitive to stimulation by hormones, > Na acetate; (b) was optimal with 0.5 M salt; (c) was guanine nucleotides, fluoride, cholera toxin, or forskolin (1not appreciably affected by the pH of the extraction 4). The lack of responsiveness of the sperm cyclase suggested buffer between pH 5.0 and 8.5;and (d)exhibited the the lack of the stimulatory guanine nucleotide regulatory greatest specific activity at the lower pH. The ex- component (Ns), and accordingly sperm particles have been tracted sperm factor could be concentrated without loss by ultrafiltration on Amicon PM-10 membranes. The used as a test system to which regulatory components from effect on adenylate cyclase of concentrated and de- other tissues might be reconstituted (3). In studies intending salted sperm extracted was inhibited 50% by various to takeadvantage of the apparent lack of modulatory subunits of the sperm adenylate cyclase, we found that combinations salts at 10 to 30 mM. adenylate The effects of the sperm factor to activate platelet of sperm enzyme preparations with brain or platelet cyclases (4, 5) resulted in substantially greater than additive adenylate cyclase, to block its inhibition via the aadrenoceptor, and to block inhibition of stimulated activities. The superadditivity which results from such reconforms of the enzyme by stable guaninenucleotides were stitutions, although suggestive of component reconstitution prevented by protease inhibitors. A 50% reduction in to sperm, was found to be due to thecontribution from sperm the sperm factor’s activationof platelet adenylate cy- of completely unrelated factor(s) (4,5). Thefactor present in clase was caused by 30 nM soybean trypsin inhibitor, sperm particles was found to activate both particulate and 30 nM aa-macroglobulin, 300 nM leupeptin, 1 p~ anti- solubilized adenylate cyclases from rat brain (4) and to actibenzamidine. Up to vate andto alter themetal-ion affinity of the adenylate cyclase pain, 15 p~ aprotinin, and 100 ~ L M 3 mM phenylmethanesulfonyl fluoride was withoutef- from platelets in aguanine nucleotide-dependent manner (5). fect on activation of the platelet cyclase by the sperm In the presentreport we describe the conditions for the factor. The effectsof the sperm factor persistedafter extraction of the activating factor from bovine sperm partiits removal by the washingof pretreated plateletmem- cles, and we present evidence that theextracted sperm factor branes and after its inactivation by the subsequent is a protease. addition of leupeptin. The data strongly support the conclusion that the bovine sperm factor is a trypsinMATERIALS AND METHODS like protease. Preparation of Human Platelet Membranes-Membranes from hua-Chymotrypsin, trypsin, and sperm acrosin were comparably effective in stimulating the platelet ade- man platelets were prepared as previously described (6)with 5 mM present throughout the membrane preparation procedure. nylate cyclase 5-to %fold, with concentrations elicit- EDTA Sperm Particles and SpermFactor-Bovine sperm, obtained either
* This work was supported by the Deutsche Forschungsgemeinschaft and a grant from the National Institutes of Health (AM 18185 to R. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. § To whom reprint requests should be sent. Recipient of an Alexander von Humboldt Foundation Fellowship on sabbatical leave to Heidelberg University.
as fresh ejaculate or washed from epididymides, were diluted with about 4 volumes of buffer containing 150 mM NaCl, 10 mM HEPES,’ pH 7.0, maintained at about 24 “C. The diluted sperm were then centrifuged for 10 min at 700 X g at room temperature. The supernatant fraction was discarded and the pelleted sperm were resusThe abbreviations used are: HEPES, 4-(2-hy&oxyethyl)-l-piperazineethanesulfonate; CAMP, adenosine3’5’-monophosphate; Gpp(NH)p,guanyl-5”yl (&y-imino)diphosphate; GTP+, guanosine 5’-O-(thiotriphosphate).
114
Extraction of the Adenylate Cyclase-activatingSperm Protease
115
pended inthe same bufferand recentrifuged. The twice-washed sperm were then frozen in small aliquots in liquid nitrogenand were stored at -85 "C. Frozen sperm were thawed withthe addition of 5 volumes of 10 mM triethanolamine HCl, pH 7.4, and were homogenized with a motor-driven Teflon-glass homogenizer. The particulate material was collected by centrifugation for 10 min at 30,000X g at 4 "C. The sperm particles were either resuspended in 10 mM triethanolamine, sperm factor pH 7.4, to be used for activation of adenylate cyclase or resuspended in an extraction buffer (see legend to Fig. 1).The supernatant fraction P \ ~ ~ L N H ~ H c o ~ resulting from the extraction was used as the sperm factor for acti. \ vation of adenylate cyclase. Adenylate Cyclase Assay-Adenylate cyclase activities were determined with a reaction mixture containing 50 mM triethanolamine HCl, pH 7.4, 1 mM 3-isobutyl-l-methylxanthine, 1 mM dithiothreitol, 0.1 mM CAMP, 0.1mM ethylene glycol his(@-aminoethyl ether)-N,N, N'N' tetraacetic acid, 5 mM creatine phosphate, creatine kinase (400 pg/ml), bovine serum albumin (2 mg/ml), 50 pM ATP, 2 mM MgC12, and [ C K ~ ~ P J(2 A TtoP5 X lo5 cpm) in a volumeof100pl. Platelet particles were incubated with the sperm factor for 15 min at 30 "C with a cyclase reaction mixture complete except for [cY-~'P]ATP. At was added, and the formation the end of this incubation [CK-~~PIATP 0.2 0.5 1.0 of [32P]cAMPwas determined after a further incubation for 10 min salt (MI at 30 "C. Cyclase reactions were terminated by precipitation with ZnC03, and [32P]cAMPwas isolatedaspreviouslydescribed (7). FIG. 1. Concentration-dependent extraction of an adenylAdenylate cyclase activities are given on the basis of platelet mem- ate cyclase activating factor from bovine sperm particles by brane protein. Protein was determined according to Lowry et al. (8) NaCl and NH,HCOs. Previously washed and frozen bovine sperm with human serum albumin as standard. Experimentswere performed (200 pl; -10s cells/ml) were aliquoted into Eppendorf tubes, were in triplicate with an intra-assay variation of less than 5% of the diluted with 1 ml of 10 mM triethanolamine HCl, pH 7.5, and were means and were repeated at least twice with results comparable to centrifuged at 30,000 X g for 10min. The resultingpellets were those reported here. resuspended in solutions containing 10 mM triethanolamine HCl, 2 Materials-[a-3ZP]ATP was prepared enzymatically as described mM EDTA, and either NaCl or NH4HC03at the indicated concenby Walseth and Johnson (9). Carrier-free (32P)phosphoricacid was trations. The mixtures were then mixed at room temperature for 5 purchased at the highest concentration available fromNew England min following which they were centrifuged at 30,000 X g for 10 min. Nuclear. ATP, GTP, Gpp(NH)p,GTP+, CAMP, creatine phosphate, Five-pl aliquots of the resulting supernatant fractions (supernates) creatine kinase, a*-macroglobulin, and soybean trypsin inhibitorwere were assayed for activating activity.The pellets were resuspended in obtained fromBoehringerMannheim.Epinephrine,phenylmeth1 ml of 10 mM triethanolamine HCI, pH 7.5, were recentrifuged, were anesulfonyl fluoride, benzamidine, aprotinin (A-4529), antipain, tryp- resuspended again in 0.5 ml of 10 mM triethanolamine HCl,pH 7.5, sin, and a-chymotrypsinwere from Sigma. Leupeptin was purchased and then were used for assay of cyclase-activating activity. Protein from Peninsula. Purified boar acrosinwas a generous gift of Dr. W. was determined in the supernatants (upperpanel). Muller-Ester1 of the Department of Clinical Chemistry and Biochemistry, University of Munich. Purified N. protein, preactivated with Gpp(NH)p,was a generous gift of Dr. T. Pfeuffer of the Department curred with 0.5 M salt'. Higher concentrationsof salt (up to2 of Physiological Chemistry, Universityof Wurzburg. All other mate- M) extracted neither additional protein (Fig. 1, upper panel) rials were obtained at the highest purity available from commercial nor additional factor. The slight decrease in activity shown sources. here with 1 M salt, relative to activity measured with extract of 0.5 M salt, was not seen when 1-pl or 2-pl aliquots of the RESULTS sperm extract were tested and are due to thepresence in the extract of an inhibitory activity. In the experiments repreSalt Requirements for Extraction of the Sperm FactorSperm particles exhibit an adenylatecyclase activity of 0.07 sented inFig. 1, no attemptwas made t o adjust the pHof the to 0.1 nmol of cAMP(min .mg protein)", with Mn*+/MnATP extraction buffer; the buffer with NaCl was pH 7.5 whereas as substrates, which is unaffectedby prostaglandins, fluoride, that with NH,HCO, approached 8.5. In other experiments, though, where the pH of the extraction buffer was varied forskolin, or guanine nucleotides (1-4). We found that the extraction of bovine sperm particles with 1% Lubrol-PX (w/ between 5.0 and 8.5, there was no change in the amount of from thespermparticles,althoughthe v) released into a supernatant fraction(30,000 X g for 10 min) factorextracted amount of protein extracted in the supernatant fraction ina n adenylate cyclase (4.5to 5 nmol of cAMP(min.mg protein)"), whichwasequally unresponsiveto modulatory creased from 80 pg/ml at pH 5.04 to 145 pg/ml at pH 8.51. agents. When such supernatant fractions were tested for their That is, the specific activity of the stimulating activity was effects on thesolubilized brain adenylate cyclase, in contrast somewhat higher with extraction buffer of lower pH. Sperm Preparation Prior to Extraction-For the original with the superadditivity seen with sperm particles (4), no superadditivity was found. (Since the platelet adenylate cy- studies of superadditivity of sperm particles and brain adeclase is inhibited by very-low concentrations of detergents nylate cyclases, the sperm were washed from the epididymal of larger (lo), platelets could not be used for testing of detergent- ducts of bovine testes (4). Forthepreparation extracted factor.) The finding that detergent could extract quantities of factor itwas desirable to use the larger amounts of sperm available through artificial insemination facilities as adenylate cyclase, but not the factor causing the superadditivity with the brain cyclase, suggested that the factor, rather fresh ejaculates. Fresh ejaculates were either frozen immedithan being an intrinsic membrane component, might be hy- ately in liquid nitrogen or were washed in buffer containing 150 mM NaCl, 10 mM HEPES, pH 7.0, prior to freezing in drophilic and, therefore,possibly released by salt. The effect of increasingconcentrations of NaCland liquid nitrogen. As can be seen in Fig. 2, only sperm which NHdHC03 to release into a 30,000 X g supernatant fraction had been previously washed free of other semen components an adenylate cyclase-stimulating activity is shown in Fig. 1. and had been frozen were capable of yielding active factor The corresponding decrease in stimulatory capacity of the upon subsequent salt extraction. particulatefraction is also shown. Optimalextraction ocUltrafiltration and Sensitivity to Salts andHeat-Following
I
Extraction of the Adenylate Cyckrse-activating Sperm Protease
116 901
.
P"
.
TABLEI Effects of various salts on adenylate cyclase activated by the sperm extract Sperm factor was derived as described for Fig. 2 from washed-then-
frozen sperm. The extract was concentrated and desalted as described in the text. Sperm Addition Basal extract
nmol (IO min mg protein)"
Experiment I None
\
, 2
(frozen semen1 ,
r,
6
,
,
8
,
2.77 ? 0.09 ( n = 8) 2.39 0.61 2.30 0.74 1.41 0.06 2.20 0.63
0.87 0.94 0.79 0.73
6.03 3.80 1.70 0.66
10 mM NaCl 100 mM NaCl 10 mM Na acetate 100 mM Na acetate 10 mM Na phosphate 100 mM Na phosphate 10 mM NHlHCO3 100 mM NH,HCOI
\
0
0.22
,
10
Yl FIG. 2. Activating factor from sperm prepared in different ways. Freshly ejaculated bovine sperm either were frozen rapidly in liquid Nz and stored at -85 "C or were maintained at 20-25 "C until preparation of the particles several hours later. Frozen whole ejaculates were thawed and then otherwise treated as fresh sperm. Fresh sperm (typically 50 ml from 5 bulls) were diluted about 3-fold (e.g.to 145 ml) with a solution containing 150 mM NaCl, 10 mM HEPES, pH 7.0, at room temperature and were then centrifuged for 10 min at 700 X g. The turbid supernatant fraction was discarded, and thepellet fraction was resuspended in n volume equivalent to theinitial volume of the pooled ejaculate (e.g. 50 ml). The washed sperm were then either frozen in liquid Nz or were maintained at room temperature. In both cases 2OO-pl aliquots of the sperm were then diluted with 1 ml of mM triethanolamine HCl, pH 7.4, were centrifuged for 10 min at 30,000 X g, and theresulting pellets were resuspended in 500 p1 of an extraction buffer consisting of 500 mM NH,HC03, 2 mM EDTA, and 10 mM triethanolamine HC1. The mixtures were mixed for 5 min at room temperature and were recentrifuged for 10 min at 30,000 X g. The resulting supernatant fraction was assayed for cyclase activating activity. Compared are NH4HC03extracts of sperm derived from quickly frozen whole ejaculates, of sperm washed but not frozen before extraction, and of sperm washed free of other semen components and then frozen before extraction. For comparison, activation of the cyclase by particles of washed-then-frozen sperm is also shown.
Experiment I1 None 24 mM(N&)&% 80 mM (N&)sSOI 240 mM (NHd2SO4
phosphate resulted in cyclase activities below basal activity (Table I). The inhibitory effect of these salts may be due either to a blockade of the effect of the sperm factor to activate the cyclase or to an enhanced sensitivity of the cyclase to inhibition by salt upon activation by the sperm factor. In earlier studies with sperm particles, we had noted that the cyclase-activating capacity of the particles was fully stable to heating for 10 min at 58 "C (4). In similar experiments with the extracted and desalted factor, we observed 50% loss of activity with exposure at 60 "C for 3 min. This half-time was not altered by the readdition of 40 mM NaC1. Thus, the sperm factor, once removed from the particulate matrix, exhibited a significant increase in sensitivity to heat. Relationship betweenActivation andProtein Concentration-The quantifying of the amount of activating factor present in the sperm extract is made difficult by the biphasic character of the factor's concentration curve as well as by the dependence of the degree of activation on the amount of platelet membrane protein used in the cyclase assay (Fig. 3). At low amounts of platelet protein(e.g. 5.2 pg/100 pl) maximal activation of the adenylate cyclase by the sperm extract was more than three times the activation seen at higher (e.g. 46.5 extraction of the cyclase-activating factor from sperm, the pg/100 pl) amounts of platelet protein. Moreover, at the low extract was concentrated 10- to 15-fold by ultrafiltration on concentrations of platelet protein the cyclase was more senAmicon PM-10 membranes. In no experiment could we detect sitive to inhibition (relative to maximal activation) by eleactivator in the filtrate, and the amount of activator in the vated amounts of the sperm extract. concentrate roughly accounted for all of that in the initial Effects of Protease Inhibitors-The data shown in Figs. 4 extract. The saltin the concentrated extractswas then essen- and 5indicate that theactivation of platelet adenylate cyclase tially completely removed by passage over a column of Bio- by the sperm factor couldbeblockedby several protease Rad P-2 gel. The desalted extract was then tested for its inhibitors and strongly suggest that the factor is a protease. thermal stability and for the effects of salt on its capacity to A 50% reduction in the stimulatory effect of the sperm factor activate adenylate cyclase. was obtained with approximately 30 nM (-0.6 pg/ml) soybean The activity of the platelet adenylate cyclase in the presence trypsin inhibitor, 300 nM (0.14 pg/ml) leupeptin, or 100 p M of desalted sperm factor was significantly reduced by several benzamidine (Fig. 4), andwith approximately 30 nM (-22 pg/ salts(TableI). In other experiments we observed a 50% ml) cup-macroglobulin, 1 p M antipain, or 15 p M (-100 pg/ml) reduction in activity in thepresence of the sperm extract with aprotinin (Fig. 5). High concentrations of benzamidine (>1 typically 20 to 30 mM NaCl, Na acetate,ammonium bicarbon- mM) oraprotinin (>lo0 pM) reduced control activity. In ate, and ammonium sulfate. At these concentrations none of contrast with the blocking action of these protease inhibitors, these salts showed significant effects on control enzyme ac- phenylmethanesulfonyl fluoride, at concentrations up to 3 tivity. However, for sodium phosphate 50% inhibition was mM, did not affect the sperm factor-induced increase in plateseen at 10 mM, and concentrations of 80 to 240 mM sodium let adenylate cyclase activity (data not shown).
Extraction ofAdenylate the
Cyclase-activating Sperm Protease
117
PLATELET PROTEIN
2
L
6 0 10 SPERM EXTRACT (yll
12
1L
FIG. 3. Dependence of adenylate cyclase activation on concentrations of platelet membranes and of sperm factor. The sperm factor was extracted from washed sperm particles with 2 M NaCI, 10 mM triethanolamine HCl, and 2 mM EDTA. This extract was desalted by passage over a Bio-Rad P-2 gel column, equilibrated and run with 20 mM triethanolamine HCl, pH 7.4. The pooled desalted extract was then concentrated about 10-fold by ultrafiltration with an Amicon PM-10 membrane. Varying amounts of this concentrate were then used for assays with adenylate cyclase of platelet membranes at theindicated amounts of membrane protein/ 100-pl reaction volume. I
3
SPERM EXTRACT
4 """""-
"--"".
"7-%
0
-9
-3
-8 -7 -6 -5 -4 I N H I B I T O R CONCENTRATION ( l o g ( M ) )
FIG.5. Inhibition by various protease inhibitors of sperm factor-enhanced stimulation of platelet adenylate cyclase. Adenylate cyclase activity of human platelet membranes (5.3 pg protein/ as tube) was determined with 100 pM MgATP and 2 mMM&12 substrate in the absence (open symbols) or presence (closed symbols) of sperm extract (2.7 pgprotein/tube) inthe absence (X, *) or presence of a2-macroglobulin (0,O), antipain (A, A), or aprotinin (0, U). GTPyS (10p ~ was ) present under all conditions.
\
\ I
\ \ I
\
\ \ .\
soybean
I
SOYBEAN INHIBITOR
\
\
5
~ l ,
\
5 ~~
L
~ ~~
~ ~~
leupeptin
~
~ ~
g / m~ l l \ . ~~~ ~b 3 p~ ~
1.6
~ inhibitor
~
h
\CONTROL
FIG. 4. Inhibition by various protease inhibitors of sperm factor-enhanced stimulation of adenylate cyclase. Adenylate cyclase activity of human platelet membranes (7.4 pg protein/tube) was determined with 100 p~ ATP and 2 mM MgC12 as substrate in the absence (open symbols) or presence (closed symbols) of sperm extract (2.4pg protein/tube) in the absence (X, *) or presence of soybean trypsin inhibitor (0,O) leupeptin (A, A),or benzamidine (0, U).GTP-yS (10 p ~ was ) present under all conditions.
Since the response of adenylate cyclase to increasing concentrations of the sperm factor is biphasic, stimulatory at low concentrations, and, relative to the peak activity, inhibitory at higher concentrations,and since sperm are known to contain numerous proteases, the sensitivity of the stimulatory and inhibitory phases to protease inhibitors was tested (Fig. 6).Concentrations of leupeptin (0.3 pg/rnl), soybean trypsin
01
1.0
10
1
SPERM EXTRACT 1pll
FIG.6. Effect of severalprotease inhibitors on the sensitivity of the platelet adenylate cyclase to stimulation and inhibition by a sperm extract. Human platelet membranes (8.6 pg protein/tube) were incubated with increasing amounts of a bovine sperm extract (14.4 pg proteinlpl) in the absence (control, solid line) or presence of leupeptin (0.3 pg/ml; 650 nM; O), soybean trypsin ; Adenylinhibitor (10 pg/ml; 500 p~ V),or benzamidine (300 p ~0). ate cyclase activities were determined with 100 p~ MgATP, 2 mM MgClz, and 10 p~ GTPyS.
Extraction of the Adenylate Cyclase-activating Sperm Protease
118
inhibitor (10 pg/ml), and benzamidine (300 p ~ blocked ) the inhibitory effect of the sperm factor and still allowed stimulation of the platelet adenylate cyclase by the sperm extract. The inhibitory effect was not seen with either the trypsin inhibitor or benzamidine and was much diminished with leupeptin at amounts of sperm extract up to10 pl. The entire activation curve for the sperm factor was shifted to the right by about an order of magnitude. In other experiments we found that the inhibitory effect of the sperm factor was blocked at lower concentrations of these inhibitors than was the stimulatory effect. Although it is not clear from these observations whether the stimulatory and inhibitory effects of the sperm extractare due to the same or to different proteases, it is of practical value that small amounts of protease inhibitor can be used to study preferentially the stimulatory effects of the sperm extract, andlarger amounts of one of these protease inhibitors (but not phenylmethanesulfonyl fluoride) can be included to protect the cyclase against proteolytic stimulation as well. If the guanine nucleotide-dependent stimulation of the platelet adenylate cyclase is due to a protease, then other effects we previously reported and ascribed to thesame factor should also beblockedby protease inhibitors. The sperm factor enhanced prostaglandin El-stimulated adenylate cyclase activity in plateletsand prevented its inhibition by epinephrine (5). Both actions of the sperm factor were blocked by leupeptin as was the activation of the GTPyS-stimulated enzyme. The effects of the sperm factor on prostaglandin Elstimulated adenylate cyclase and on its inhibition by epinephrine are shown in Table 11. The 60% inhibition of the prostaglandin E,-stimulated cyclase seen with 30 p~ epinephrine was completely lost with the sperm factor but was retained in the presence of leupeptin (10 pg/ml). The above data and previously reported observations are TABLEI1 Influence of the bovine sperm factorand leupeptin on inhibitionof human platelet adenylatecyclase by epinephrine Adenylate cyclase activity of human platelet membranes (36 pg protein/tube) was determined as described under “Materials and Methods” without and with epinephrine (30 p ~ in) the absence and presence of leupeptin (10 pglml), bovine sperm factor (46 ng protein/ tube),or their combination. Prostaglandin El (1 phf) and GTP (1p M ) were present under each condition. Numbers in parentheses indicate the per cent inhibition by epinephrine. Addition
Control
Epinephrine
pmol CAMP(rnin X m&“
None
109269 276 433 256
Leupeptin (10 pg/ml) Sperm factor (46 ng) Sperm factorplus leupeptin
(60) 110 (60) 432 (0) 104 (60)
TABLE111 Interactions of N. protein, Gpp(NH)p, and the bovine sperm factor on the adenylate cyclase of human platelets Adenylate cyclase activity of human platelet membranes (48 pg protein/tube) was determined as describedunder “Materials and Methods” in the absence and presence of Gpp(NH)p,100 p ~leupep; tin, 10 pg/ml; N. protein, 12 ng/tube; and bovine sperm factor, 50 ng protein/tube. Control Addition
Basal
Gpp(NH)p
Sperm factor Basal
Gpp(NH)p
pmol CAMP(min x m&’
None
Leupeptin (10 pg/ml) N. protein (120 ng/ml) N. protein + leupeptin
7.0 10.0 149 153
39.2 39.3 65.3 65.2
16.8 8.0 297 151
405 38.0 566 63.0
consistent with the idea that the sperm factor alters the adenylate cyclase system by causing a functional blockade of the inhibitory guanine nucleotide-dependent coupling component Ni. In an initial approach to this question we investigated the influence of the sperm factor on inhibition of the humanplatelet adenylate cyclase by stable GTP analogs. Whereas basal platelet adenylate cyclase activity is increased by stable GTP analogs, the enzyme can be inhibited by these nucleotides when it is stimulated by other agents, such as prostaglandin El (11) forskolin (12), or by preactivated N. protein (Table 111).As shown in Table 111,100 ~ L MGpp(NH)p increased basal activity about &fold, whereas the enzyme stimulated by preactivated N. protein (120 ng/ml) was inhibited by Gpp(NH)p about 60%. Under the assay conditions used, i.e. 15 min of preincubation a t 30 “C,both the N. protein-stimulated and theGpp(NH)p-inhibited enzymes exhibited linear time courses for cAMP formation for at least 20 min. The Gpp(NH)p-induced inhibition was also observed when the platelet enzymewas preactivated byN. protein (data not shown). Addition of the sperm factor (Table 111) increased the stimulation by Gpp(NH)p about 10-foldand the stimulation byN. protein about %fold. The sperm factor caused a loss of the Gpp(NH)p-induced inhibition of the N. protein-stimulated cyclase activity and resulted in Gpp(NH)p causing an even greater activation. Observations similar to those made here with Gpp(NH)p were also seen with GTPyS (data not shown). Each of these respective actions of the sperm factor was blocked by leupeptin (10 pg/ml), which had no effect on control activities. These several observations support the suggestion that the effects of the sperm factor may be dueto a common mechanism of action on the adenylate cyclase involving proteolysis of one of its constitutive subunits. Reuersibility of the Effects of the Sperm Factor-If the effects of the sperm factor are due to a protease, it would be expected that its effects on adenylate cyclase would be irreversible. Experiments were designedto approach this question in two ways. First, since leupeptin was found to prevent the effects of the sperm factor to enhance Gpp(NH)p-stimulated adenylate cyclase (Figs. 4 and 6), leupeptin was added to the assay after full activation of the cyclase by the factor. When added after the sperm factor, leupeptin was found not to affect the rate of cAMP formation. This lack of return of the rate of cAMP formation to that seen with Gpp(NH)p alone is consistent with a persistent activation of adenylate cyclase. In a second series of experiments platelet membranes were exposed to the factor and the factor was then removed by washing with sequential centrifugation and resuspension steps, following which the efficacy of GTPyS and the sperm factor to stimulate the cyclase were retested (Table IV). In the experiment shown here, the sperm factor increased basal activity about %fold and increased GTPyS-stimulated activity about 10-fold.However,following exposure of platelet membranes to the sperm factor in a 15-min preincubation at 30 “C,the subsequent addition of the factor elicited no further increase in either basal orGTPyS-stimulated activity. In both of these cases the preincubation with the sperm factor resulted in activities comparable to those seen when membranes were preincubated without the factor but to which it was subsequently added in the assay. Thus, the full capacity to respond to the sperm factor was not impaired by the preincubation, and the preincubation with the sperm factor resulted in an adenylate cyclase whichexhibited the persistent influence of it. Important in the experiment shown in Table IV was the condition that the relationship between the amount of the
Extraction ofAdenylate the
Cyclase-activating Sperm Protease
TABLE IV Persistence of the stimulatory effect of the sperm factor Platelet membranes were preincubated for 15 min at 30 "C without or with 1 p1 of sperm extract and with a complete cyclase reaction mixture except for [w3'P]ATP and GTPyS. Thispreincubation was stopped by the addition of 2 volumes of ice-cold 10 mM triethanolamine HCl, pH 7.4, and 5 mM EDTA. The membranes were then centrifuged at 30,000 X g for 10 min and were resuspended in the same buffer. This washing of the membranes was repeated twice more. The membranes were resuspended in 10 mM triethanolamine HCl, pH 7.4, and were then incubated at 30 "C for 15 min with a complete cyclase reaction mixture, except for [w3'P]ATP, with 10 p~ GTP+ or 1 pl of sperm extract as indicated. [cY-~'P]ATPwas then added, and theformation of [32P]cAMPwas measured after an additional incubation for 10 min at 30 "C.
trypsin (Ag)
0
0,05
I'
1.0 0
119 chymotrypsin ( p g ) 0.5 1.0
I'
Additions to preincubations
Additions to incubation
:;:7
None
pmol CAMP fmin
X
mgl"
None13.1 extract Sperm (1pl) GTPyS (10p M ) Sperm extract + GTPrS
6.9 12.8
409
11.3
39.0 419 403
sperm factor and the amount of platelet membrane protein used in the preincubation was the same as that found to be optimal in theadenylate cyclase assay.That this relationship is important was evident from Fig. 3. In otherexperiments in which a lesser amount of the sperm factor relative to membrane protein was used in the preincubation a partial reversibility of the action of the sperm factor was noted. That is, the preincubation of platelet membranes with a less than optimal amount of the sperm factor, followed by its removal by washing with centrifugation and resuspension steps, resulted in an adenylate cyclase preparation which did not demonstrate the full effect of the protease and which could be subsequently further stimulated by the addition of the sperm protease to thecyclase assay.These observations would be consistent with the actions of a protease if the substrate protein on which it acts is either partially inaccessible and mobile or exists in alarge excessin themembrane relative to other adenylate cyclase components. Comparison of the Sperm Factor with Other ProteasesSperm are known to contain a number of proteases, a predominant one being acrosin (13), though effects of acrosin on adenylate cyclase have not been reported. However, proteolytic activation of adenylate cyclase has been reported for a number of proteases and with enzymesfrom a variety of sources (e.g. 14-19). Hanoune et al. (14) reported a 2- to 3fold increase in basal and hormone- or fluoride-stimulated adenylate cyclase activities of rat liver membranes by crude preparations of collagenase. These effects could be mimicked by elastase. More recently these workers and others reported an uncoupling of a-adrenergic receptor-mediated inhibition of the platelet adenylate cyclase by treatment with a-chymotrypsin (18)or trypsin (19). However,in studies of the pigeon erythrocyte adenylate cyclase, Marshak and Neer (16) observed a biphasic stimulatory effect of a-chymotrypsin but only observed inhibition with trypsin. It was thus important to compare the actions of the sperm factor on adenylate cyclase with the effects of other proteases, and particularly with trypsin and acrosin. In Fig. 7 the stimulatory effects of acrosin, a-chymotrypsin, and trypsin are compared with the effect of the extracted sperm factor. Trypsin, a-chymotrypsin, and acrosin were comparably effective, each stimulating the cyclase 5- to 8-
ocrosin ( A g )
sperm extract
(AI)
FIG.7. Comparison of several proteases in enhancing GTPyS-stimulated adenylate cyclase activity. Human platelet membranes (8 pg protein/tube) were incubated with the indicated amounts of the various proteases, and adenylate cyclase activities were determined. GTPyS (100p ~was ) present undereach condition. The protein concentration of the crude sperm extract was 7.14 pg/ ml. Note that theabscissas for the several proteases differ.
incubation time(minl FIG.8. Comparison of time courses for activation of platelet adenylate cyclase by trypsin and the extracted sperm protease. Complete cyclase reaction mixtures were thermally equilibrated at 30 'C, and reactions for the indicated times were initiated by the addition of human platelet membranes (10pg protein/tube). CAMP formation was assayed without (0) or with the indicated concentrations of trypsin (doshed lines) or sperm extract (7.14pg protein/ml; solid lines). GTP+ (10 PM) was present under each condition.
fold. Of these enzymes trypsin was about an order of magnitude more potent than either a-chymotrypsin or acrosin. Maximally stimulatory effects were seen with 20 ng of trypsin, but with 200 ng of a-chymotrypsin or acrosin. By comparison, the crude sperm factor increased the GTPyS-stimulated adenylate cyclase activity about 25-fold, witha maximally stimulatory amount of 700 ng. While direct comparisons of po-
120
Extraction of the Adenylate Cyclase-activating Sperm Protease
tency cannot be made between the crude sperm factor and the otherpurified proteases, it isnonetheless evident that the sperm factor was appreciably more effective than these other proteases in elevating adenylate cyclase activity. Since trypsinand thesperm factor areproteolytic enzymes, the relationship of the amount of enzyme to the amount of substrate protein and the time of incubation become important considerations for making any comparisons of their effectiveness. To determine whether the lesser effectiveness of trypsin shown in Fig. 7 might have been due to a less than optimal balance between protein concentrations and incubation times, the effect of trypsin and the sperm factor were compared as a function of their time of incubation with the platelet membranes (Fig. 8). The lag phase typical of the action of the sperm factor is evident and increased from about 12 to 24 min when the amount of the sperm factor was decreased from 1.0 to 0.2 pl, yet the maximal rate of formation of cAMP was the same with both amounts of the factor. That is, not surprisingly, lesser amounts of the sperm factor achieved the same end catalytic effect on the cyclase with longer incubations as did larger amounts with shorter incubations. A similar relationship was seen with trypsin (Fig. 8). Larger amounts (50 ng) of trypsin exhibited a shorter lag phase in the activation of the cyclase than did smaller amounts (20 ng). In both cases, though, whether optimally (50 ng) stimulatory (20 ng) orinhibitorysupraoptimal amounts of trypsin were used,the rateof formatiuxmf cAMP did not approach that seen with the sperm factor with any of the incubation times. In other experiments we found that the effectiveness of trypsin and thesperm factor also were different in theirability to increase the sensitivity of adenylate cyclase to free M e . We previously reported that the adenylate cyclase of human platelets exhibited half-maximal Gpp(NH)p-stimulated activity with about 4 mM M&lz (5). The sperm factor caused a marked increase in sensitivity of the enzyme to M F , with the half-maximal concentration being reduced to approximately 0.1 mMMg2+ (5). In similar experiments with trypsin, we found that the concentration of M e required for halfmaximal activity was reduced from 5 mM to approximately 1.5 mM Mg' by treatment of the platelet adenylate cyclase with trypsin.
ties to heat. We had reported previously (4,5) that the cyclaseactivating capacity of sperm particles was stable for 10 min to heating at 58 'C. A considerably greater lability of the crude extracted factor was observed (half-time at 60 "C of 3 to 4 min). An increase in lability, though, might be expected if the factor, when bound to thesperm particles, is stabilized by other membrane-bound constituents, whereas in solution it becomes more susceptible to hydrolysis or perhaps even autoproteolysis. That the sperm factor causing the various effects on adenylate cyclase is a protease is supported by several lines of evidence. First, the dependence of the degree of activation of the recipient cyclase on both sperm extract and membrane protein concentrations, together with the observed time lag for activation by the sperm factor, would be consistent with an enzymatic action of the factor on the adenylate cyclase system. Second, several protease inhibitors, notably soybean trypsin inhibitor, leupeptin, benzamidine, az-macroglobulin, antipain, and aprotinin, but not phenylmethanesulfonyl fluoride, abolished activation of the cyclase by the sperm factor and abolished the blockade of the inhibitory actions of epinephrine and stableguanine nucleotides. Third, the effects of the sperm factor were not reversible by washing. And fourth, the protease inhibitors could not reverse the effect of the sperm factor once the cyclase had been fully activated by it. When the effects of the sperm extract were compared with the effects of proteases known either to alter adenylate cyclase activity (trypsinanda-chymotrypsin) or to be presentin sperm (acrosin), the sperm extract was found to be the most effective both in increasing adenylate cyclase activity and in increasing sensitivity of the enzyme to free M$+. Although it is notpossible to comment on the potency of the sperm extract relative to theeffects of these otherproteases, since the sperm protease described here has not been purified to homogeneity, it was clear that of the other proteases trypsin was the most potent and that tobe equipotent with trypsin the protease in the sperm extract would have to be purified 20- to 40-fold, and to be equipotent with acrosin only 2- to 4-fold. One concern was that this sperm protease might actually be acrosin. However, the difference in the magnitude of the effects of acrosin and the sperm extract, their probable differences in potency, and the differences in the procedures for their extraction argue against this. Furthermore,preliminary studDISCUSSION ies have shown that indifferent chromatography systems the sperm protease described here and acrosin could be distinIn previous studies we reported the presence in sperm particles of a factor capable of significantly activating ade- guished. Thus, the data suggest that this sperm protease and nylate cyclases from various sources (4, 5). In this report we acrosin are distinct, though not necessarily unrelated. Other have presented, first, data describing the extraction of this observations suggest further that theprotease described here cyclase-activating factor from sperm particles, and, second, is a trypsin-like serine protease. First, the previous observaevidence that this factor is a protease. The data support the tion that the activity of the enzyme in sperm particles was conclusion that thefactor is released only from broken sperm not blocked by treatment of the particles with N-ethylmaleimand that itis hydrophilic in nature. Intact sperm are ineffec- ide (4, 5) would argue against its being a thiol protease. tive in stimulating adenylate cyclases of recipient membranes, Second, the profile of protease inhibitors which blocked its and the factor is released from washed sperm particles by action would be consistent with that expected for a serine salts, but not by nonionic detergents. Based on its behavior protease. And third, the preliminary observation from unpubonultrafiltration on Amicon PM-10 membranes, the ex- lished studies that thesperm protease described here catalyzes tracted factor exhibited a molecular mass of greater than reactions with synthetic trypsin substrateswould further suproughly 10,000 daltons. The extracted factor exhibited most port this suggestion. Inasmuch as thesperm protease stimulates various adenylof the properties of the particle-bound factor in that it stimate cyclases by a mechanism which apparently does not inulated adenylate cyclase in a guanine nucleotide-dependent manner, prevented inhibition of the platelet cyclase by epi- volve an effect on the cyclase catalytic unit, nor on the N. inhibitory hormone nephrine, prevented inhibition of activated adenylate cyclases protein, nor oneitherstimulatoryor receptors (e.g. 8- or a2-adrenoceptors)but blocked the actions by the stable GTP analogs Gpp(NH)p or GTPrS and increased the sensitivity of adenylate cyclase to free M$+. In of inhibitory hormones and of stable GTP analogs and inhibcontrast with these similarities inthe actions of the extracted ited the GTPase activity associated with the effects of inhiband particle-bound factor is the difference in their sensitivi- itory hormones on adenylate cyclase, the likely site of action
Extraction of the Adenylate Cyclase-activating Sperm Protease
121
5. Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983)Biochim. Biophy~.Acta 756,369-375 6. Jakobs, K. H., Lasch, P., Minuth, M., Aktories, K., and Schultz, G. (1982)J. Biol. Chem. 257, 2829-2833 7. Jakobs, K. H., Saur, W., and Schultz, G. (1976)J. Cyclic Nucleotide Res. 2,381-392 8. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Bwl. Chem. 193,265-275 9. Walseth, T. F., and Johnson, R. A. (1979)Biochim. Biophys. Acta 562,ll-31 10. Jakobs, K. H.,Saur, W., and Johnson, R. A. (1979)Biochim. Biophys. Acta 583,409-421 11. Tsai, B. S., and Lefkowitz, R. J. (1979)Biochim. Biophys. Acta Acknowledgments-We would like to express our sincere appreci587,28-41 ation for the superb technicalassistance of Gabriele Gabel, Christina 12. Jakobs, K. H., and Aktories, K. (1983)Biochim. Biophys. Acta Stannek, Linda White, and Joseph A. Awad. We would also like to 732,352-358 thank Dr. H. Bostedt of the Veterinary School, Giessen, Dr. F’iirstel, 13. Muller-Esterl, W., and Fritz, H. (1981)Methods E n z y m l . 80, of the Besamungstation Darmstadt, Griesheim, and Drs. David Gar621-632 bers and Thomas Noland of the Howard Hughes Medical Institute, 14. Hanoune, J., Stengel, D., Lacombe, M.-L., Feldmann, G., and Vanderbilt University, Nashville, Tennessee, for their help in obtainCoudrier, E. (1977)J. Bwl. Chem. 252,2039-2045 ing the bovine sperm. 15. Yamamura, H., Lad, P. M., and Rodbell, M. (1977)J. Biol. Chem. 252,7964-7966 REFERENCES 16. Marshak, D. R., and Neer, E. J. (1980)J. Biol. Chem. 255,47811. Gray, J. P., Drummond, G. I., Luk, D.W. T., Hardman, J. G., 4785 (1980)J. BWL Chem. 256,7716-7721 and Sutherland, E. W. (1976)Arch. Biochem. Bwphys. 172, 18. Ferry, N., Adnot, S., Borsodi, A., Lacombe, M.-L., Guellaen, G., 20-30 and Hanoune, J. (1982)Biochem. Biophys. Res. Commun. 108, 2. Garbers, D. L., and Kopf, G. S. (1980)Adv. Cyclic Nucleotide Res. 708-714 13,251-306 3. Stengel, D., and Hanoune, J. (1981)J. Biol. Chem. 256, 5394- 19. Stiles, G. L., and Lefkowitz, R. J. (1982)J. Biol. Chem. 267, 6287-6291 5398 4. Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G. (1983) 20. Deleted in proof 21. Combest, W.L., and Johnson, R. A. (1983)Arch. Biochem. FEBS Lett. 152,ll-16 Biophys. 225,916-927 ‘ c f . K. H. Jakobs, R. A. Johnson, M. Minuth, G. Schultz, Eur. J. 22. Katada, T., Bokoch, G. M., Smigel, M. D., Ui, M., and Gilman, A. G. (1984)J. Biol. Chem. 259,3586-3595 Biochem., submitted for publication.
of the protease would be on the guanine nucleotide inhibitory component, Ni.2 Consequently, we have named this sperm protease ninhibin. It remains to be established whether ninhibin acts primarily on theai, 8, or some other subunit of Ni. However, in view of the processes involved in the regulation of adenylate cyclase (21) and the proposed mechanisms by which ~yi,an,and 8 subunits of N. and Ni are thought to regulate the enzyme’s catalytic activity (22), we would predict that the ai subunit of Ni will be found to be the preferential target of ninhibin.
