Ca2* Stimulation of Rat Liver Mitochondrial Glycerophosphate ...

THEJOURNAL

OB ~ 1 0 L O C I C A LCHEMISTRY

Vol. 256.No. 24, Issue of December 25. pp. 12767-12i71. 1981 Printed

in (1.S.A.

Ca2*Stimulation of Rat Liver Mitochondrial Glycerophosphate

Dehydrogenase* (Received for publication, July 28, 1981)

M a r y Ellen Wernette$, Raymond S. Ochs, and Henry A. Lardy From the Institute for Enzyme Research and theDepartment of Biochemistry, Unioersity of Wisconsin, Madison, Wisconsin 53706

Ca2+stimulates rat liver mitochondrial glycerophosphate dehydrogenase activity at low, but not high, concentrations of itssubstrate, glycerophosphate. The midpoint for activation by Ca" is about lo" M. The Ca2+effect was duplicated by Sr2+,but not by M$+. Hyperthyroidism increased total glycerophosphate dehydrogenase activity 12-fold, but did not significantly affect the K, for glycerophosphate nor theresponse to Ca". Therefore, the enzyme from hyperthyroid animals was used for subsequent studies. The Ca2+sensitivity was retained in Triton-solubilized preparations of glycerophosphate dehydrogenase from sonic particles of hyperthyroid rat liver mitochondria. Phenothiazine drugs, chlorpromazine and triffuoperazine, inhibit the partial stimulation of the solubilized enzyme by subsaturating Ca" concentrations, but not the maximal stimulation achieved at saturating (20 PM) Caz+.Exogenous bovine brain calmodulin did not alter the response to Ca" (at any Ca2+concentration). After solubilization and hydrophobic column chromatography, electrophoresis resolved the inner membrane fraction into severaldiscrete protein bands. A single band was obtained upon activity staining, and the activity of this band was also stimulated by Ca2+. The resultsdemonstrate that Ca" stimulates rat liver mitochondrial glycerophosphate dehydrogenase through a decrease in K,. Although it is stili possible that a tightly associated calcium-binding protein is involved in Ca2' activation, there is no evidence that calmodulin fills this role. Regardlese of its exact mechanism, ea2+stimulation of this enzyme links the augmented cytosolic Ca2+subsequent to catecholamine action onthe hepatocyte to the stimulated reducing equivalent transfer through the glycerophosphate cycle.

changes in the cytosolic Ca2' concentration. Ca2+stimulation of mitochondrial glycerophosphate dehydrogenase has been demonstrated for lung (6) and insect flight muscles (7). However, it has been reported that Ca2+ does not stimulate the enzyme from Liver mitochondria (8). Although it is estabIished that long term thyroid hormone treatment greatly increases the total activity of glycerophosphate dehydrogenase (9), the K,,, for glycerophosphate has also been reported to increase (10). In this report, we have re-examined the question of Ca2' control of glycerophosphate dehydrogenase from liver mitochondria of euthyroid and hyperthyroid rats. Preliminary reports of this study have been presented (11,12). EXPERIMENTALPROCEDURES

Materials-Triton X-100, iodonitrophenyltetrazolium violet (grade I), Hepes,' and digitonin were obtained from Sigma. Digitonin was recrystallized from ethanol before use. Bovine serum albumin depleted of fatty acids and triiodothyronine were from the Nutritional Biochemicals CO.Trifluoperazine and chlorpromazine were gifts from Smith-Kline and French and calmodulin (isolated from bovine brain) was donated by Drs. Frank Siege1 and Michael Smith of the University of Wisconsin. Animals-Male Sprague-Dawiey rats (200-250 g)wereallowed free access to food and water. Hyperthyroidism was induced by daily subcutaneous injections of 0.5 mg of triiodothyronine (asa solution in alkaline saline) per kg body weightfor at least 7 days prior to sacritke. Isolation ofMitochondria-Rat liver mitochondria were prepared by the method of Johnson andLardy (13) using a medium of 250 mM mannitol, 70 m~ sucrose, 3 mM Hepes, and 1 m~ EGTA, a t pH 7.4, throughout the preparation. Solubilization of Glycerophosphate Dehydrogenase-Mitoplasts were prepared by digitonin treatment of mitochondria (14).Optimal recovery was obtained at 0.15 mg of digitonin/mg of mitochondrial protein using suspensions of 50 mg of mitochondriajml plus 1 mg/ml of bovine serum albumin. Sonic particles were prepared by suspension of mitoplasts in 10 mM potassium phosphate buffer, pH 7.5, with 0.2 m~ dithiothreitol and 1 m~ EGTA, sonication at setting 6 of a Branson sonicator, and centrifugation for 45 min at 100,ooO X g.The When an excess of reducing equivalents (NADH) is present 100,oOO X g pellet was resuspended in 10 m~ potassium phosphate, 1 in the cytosol of the hepatocyte, two major shuttle systems, m~ EGTA, 0.05% Triton, 0.2 m~ dithiothreitol, and 0.02% sodium azide, pH 7.5, at a protein concentration of 15 mg/ml. The suspension the malate-aspartate and glycerophosphate shunts, transport was brought to a final Triton concentration of OB%, stirred 20 min a t them into the mitochondria (1). Of these, only the glycero- 0 "C, and centrihged 45 min at lOa,aoO X g . The supernatant of this phosphate cycle responds to an augmented cytosolic Ca2+ step contained the solubilized glycerophosphate dehydrogenase. Glycerol (35%, final concentration) was added, and the preparation was subsequent to catecholamine action (2,3). The rate-limiting step of the glycerophosphate cycle is the stored at -20 "C. Hydrophobic Affinity Column-Hydroxymethylhexamethylenemitochondrial glycerophosphate dehydrogenase (4).This endiamine-succinyl-hexamethylenediamineSepharose 4B was synthezyme is located on the outer face of the inner mitochon&id sized and used for purification of the solubilized glycerophosphate membrane (5) and, therefore, could respond directly to dehydrogenase as described by Cole et al. (15) with the inclusion of I mM EGTA in each chromatography buffer. The glycerol-stored * This research was supported by Grants AM 10,334and AM 20,678 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. ' The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperaThis article must therefore be hereby marked "advertisement" in zineethanesulfonic acid Bicine, N,N-bis(2-hydroxyethy1)glycine; accordance with 18 U.S.C. Section 1734 solely to indicate this fact. BSA, bovineserum albumin; EGTA, ethylene glycol bis(f?-aminoethyl d Predoctoral trainee, supported by National Institutes of Health ether)-N,N ,N', N'-tetraacetic acid; INT, p-iodonitrotetrazolium viProgram 5 T32 GM07215. olet; Mops, 4-morpholinepropanesuIfonicacid.

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Ca 2+ Stimulates GZycerophosphate Dehydrogenase

12768

enzyme wasdiluted IO-fold prior to application. Active fractions were concentrated under pressure (N2 gas) using a PMlO Millipore filter. Protein Assays-Protein concentration was determined by the biuret reaction using BSA as a standard (16). Solubilized samples were compared to BSA plus Triton standards.In some cases, the dyebinding protein assay (17) was used with equivalent results. Assays of Glycerophosphate Dehydrogenase Activity-For intact mitochondria, enzyme activity was determined by a minor modification of the method of Gardner (18).Mitochondria were incubated in a final volume of 0.4 ml containing 50 m~ potassium phosphate (pH 7.0), 2 m~ EGTA, 1 mM KCN, 0.2% INT, and various amounts of D,L-a-glycerophosphate.The values given for glycerophosphate concentrations refer only to the L isomer. For determinations in the presence of Ca2+,CaClz was first mixed with the EGTA, and this solution was adjusted to pH 7.0 before its addition to the assay. Usually 200 pg of mitochondrial protein from euthyroid rats or 20 pg of mitochondrial protein from hyperthyroid rats was used. The assay was linear with protein concentration up to at least 400 pg with euthyroid rats and with time up to40 min. Free Ca2+concentrations were calculated as described by PortzeN et al. (19). Mgz+and S?' were added as chloride salts and free concentrations were calculated from the apparent EGTA-binding constants of 40.1 M" and 1.56 X 10' M", respectively (20). With the Triton-solubilized glycerophosphate dehydrogenase, the same conditions as above were used except Triton X-100 was included at a final concentration of 0.0075%.Enzymatic activity was severely limited a t Triton concentrations of less than 0.005%.Since the presence of Triton interferes with the ethyl acetate extraction of the formazan, these assays were stopped with 0.6 ml of 3.2 M acetic acid and 4%Triton solution. After 10 min, the formazan was completely solubilized and absorbance at 490 nm was read directly. To obtain the extinction coefficient of the formazan product of INT in the presence of Triton, INT was titrated with dithiothreitol under alkaline conditions. This gave an €490 of 1.83 X lo4 M" cm", whi:.h was used to calculate activities for the solubilized enzyme.The pror.t:dure applied to the formazan in ethyl acetate gave the same value reported by Pennington (21), c490 = 2.01 X lo4 M-' cm", which was used in enzyme activity calculations for intact mitochondria. Polyacrylamide Gel Electrophoresis-Electrophoresis was performed by a minor modification of the method of Dewald et al. (22) with 1 m~ EDTA and 0.1% Triton X-100 present in the gels and buffers. Samples were applied in 33 m~ Tris-C1, pH 6.7, 2 m~ sodium phosphate, 0.5 m~ EDTA, 0.2 mM dithiothreitol, 10 m~ thioglycolate, and 10% glycerol.Coomassie brilliant blue wasused to stain for protein within the gels.

BO

60 c

I

4

I

-: 40

30 I

I

I

I

FIG. 2. Ca2+titrations of glycerophosphate dehydrogenase activity. Glycerophosphate concentration was 1.5mM. A , normal mitochondria; each point represents the mean of six experiments; B , solubilized glycerophosphate dehydrogenase from hyperthyroid mitochondria; the single experiment shown is representative of four separate preparations; C, hyperthyroid mitochondria; each point represents the mean of five experiments. Calcium concentrations for half-maximal stimulation are: 0.09 p~ for normal mitochondria, 0.3 PM for hyperthyroid mitochondria, and 0.6 p~ for the solubilized enzyme. Actiuity Stain for Glycerophosphate Dehydrogenase-After electrophoresis, gels were bathed 1h in 50 m~ potassium phosphate, pH 7, 1 ~ B KCN, I and either 2 mM EGTA or 2 ri EGTA plus 2 mM CaC12. Subsequently, gels were incubated at 37 "C in the complete assay cocktail used for intact mitochondria. Reaction was stopped by transfer of the gels to 10%acetic acid. Gels were scanned at 490 nm using a Gilford linear transport attachment for cylindrical gels. Relative activities were determined by integrating the peak areas. Activity determined in this manner was linear with time for up to 2 h in the presence or absence of Caz+. RESULTS

I

I

1

02

04

1 06

I/[L-a-Glycerophosphate],

I

1

08

IO

(mMf'-

FIG. 1. Effect of Ca" on the glycerophosphate dehydrogenase activity of euthyroid rat liver mitochondria.Calcium addition represents 2 p~ free Ca2+.Each point represents the mean of six experiments. Without Ca2+,Vmax= 8.6 nmol/min/mg of protein.

Stimulation of GlycerophosphateDehydrogenase in Intact Rat Liver Mitochondria by Calcium Ions-The glycerophosphate dehydrogenase of euthyroid rat liver mitochondria is stimulated by Ca2+ whenassayed at low substrate concentrations as reflected by a decrease in the K,,, for glycerophosphate from 4.5 mM to 1.3 mM (Fig. 1). A s m d butsignificant decrease of maximum velocity by Ca'' occurs as a result of inhibition of the iodonitrotetrazolium-coupled assay (23). A titration of the response to various ea'+ concentrations is shown in Fig. 2A. Half-maximal stimulation occurs at 0.09 FM free Ca2+.The effect of calcium is mimicked by Sr" but not by M$+ (Table I).' Properties of Glycerophosphate Dehydrogenase in Intact For experiments in the presence of ST2+,the calculated concentration of free EGTA was 0.5mM. Therefore, the stimulation by Sr2' does not represent displacement of the endogenous Ca2+from EGTA.

Ca2+Stimulates Glycerophospha.te Dehydrogenase

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Liver Mitochondria from Hyperthyroid Rats-Hyperthyroidism is known to induce mitochondrial glycerophosphate dehydrogenase (9). As shown in Fig. 3, a 12-fold increase in glycerophosphate dehydrogenase activity is observed in hyperthyroid rat liver mitochondria, while the values of K, for glycerophosphate in the absence and presence of Ca2+ are similar to the euthyroid values, 3.8 m~ and 1.4 m, respectively. Half-maximal response to Ca2+occurs at 0.26 pM free Ca" (Fig. 2 C ) . The hyperthyroid rat liver mitochondrial glycerophosphate dehydrogenase was used for further studies. Partial Purification of Triton-solubilized Glycerophosphate Dehydrogenase-Specific activity of the Triton-solubilized fraction of inner mitochondrial membranes from hyperthyroid rats ranged from 98-200 nmol/min/mg for four preparations. The behavior of the rat liver enzyme on the hydrophobic matrix of hydroxymethylhexamethylenediamine-succinyl-hexamethylenediamineSepharose 4B was similar to that of the rabbit muscle enzyme as described by Cole et al. (15). The activity was concentrated in a yellow band at the top of the column during application and was quickly TABLEI Efiects of divalent metal ions ongLycerophosphate dehydrogenase activity of normal rut liver mitochondria free Metal ion

ion concentration

Glycerophosphatedehydrogenase activity 1.5 mM GP"

50 m M GP

0 2

04

06

I/[L-u-Glycerophosphate],(mM)

-

FIG. 4. Effect of Ca" on solubilized glycerophosphate dehydrogenase activity. Calcium additionrepresents 20 p~ free Ca2+.

nrnol/min/mgproteinb

M

8.97 & 0.70 None 2.64 k 0.29 5.16 f 0.50' 8.12 f 0.5gd 1.9 X IO"' Ca2+ 1.4 X 2.53 k 0.27 8.56 f 0.62 Mg2' 1.9 X 4.97 2 0.45' 8.36 f 0.74d ST2+ GP, glycerophosphate. * Each value is the mean 2 standard error of eight experiments. 'p < 0.05 by comparison with controlat 1.5 m~ glycerophosphate. dp 0.05 by comparison with control at 50 m~ glycerophosphate.

TABLEI1 Effects of chlorpromazine, trifluoperazine, and calmodulin on ea2'stimulation of solubilized glycerophosphate dehydrogenase activity Glycerophosphate concentrationis 1.5 mM. Addition

Glycerophosphate dehydrogenase activity No Ca2+ 0.5 pM free 20 ~ I Mfree CaL+ Ca" added ~~~

~~

nrnol/min/mgprotein"

None 51 101 (200)' 121 (240) 41 72 (180) 105 (260) Chlorpromazine, 100 p~ 117 (240) 48 90 (190) Trifluoperazine, 50 p~ 107 (230) 72 47 (150) Trifluoperazine, 100 p~ Calmodulin, 100 ng 118 (240) 50 99 (200) Results of a single experiment which is representative of three preparations. * Values in parentheses are Ca2+-stimulatedactivity as per cent of the activity without added Ca2'.

h a .0 0)

02 I/

04

0.6

[ L-=-Glycerophosphate],

0.8

IO

(mM)" --f

FIG. 3. Effect of Caz+on the glycerophosphate dehydrogenase activity of hyperthyroid rat liver mitochondria. Calcium addition represents 2 p~ free Ca". Each point represents the mean of five experiments. Without Ca*+, V,,, = 101 nmol/min/mg of protein.

eluted with 0.3 M NaCI. Recovery ranged from 65-90% of the total activity applied with 2- to 2.5-fo'd increased specific activity. Properties of Triton-solubilized Glycerophosphate Dehydrogenase-The K,,, for glycerophosphate of the solubilized enzyme is higher than for the intact hyperthyroid mitochondria and ranged from 8.0-12.8 mM in the absence of Ca2+.An example of the Ca" effect onthe solubilized enzyme is shown in Fig.4. A 2- to 3-fold decrease in the K, for glycerophosphate was generally obtained in the presence of20 free Ca2+.A typical titration of the stimulation by Ca2+is presented in Fig. 2B. Ca2+ concentrations for half-maximal response ranged from 0.40-1.26 p~ free Ca". Effect of Exogenous Calmodulin and of PhenothiazinesThe possible involvement of ealmodulin in the Ca2+stimulation of glycerophosphate dehydrogenase was examined with two phenothiazines, trifluoperazine and chlorpromazine, which are known to inhibit calmoddin-dependent Ca2+responses (24), and by addition of exogenous purifiedcalmodulin. Neither trifluoperazine, chlorpromazine, nor calmodulin

Ca 2+ Stimulates Glycerophosphate Dehydrogenase

12770

-

a

b

affected the ability of Ca2' to stimulate glycerophosphate dehydrogenase activity maximally (Table 11). The phenothiazines did inhibit partially the activation of glycerophosphate dehydrogenaseactivity by subsaturatingconcentrations of Ca". This corresponds to an increase of the concentrationof Ca2' requiredtoachieve half-maximal stimulation in the presence of these compounds. No consistent effect of trifluoperazine,chlorpromazine, or calmodulincouldbedemonstrated for intact mitochondria when glycerophosphate dehydrogenase was assayed in the presenceof subsaturating or saturating Ca" concentrations (data not shown). Ca2+Stimulation in Polyacrylamide Gels-To distinguish between a Ca2+ stimulation directly at the enzyme or a requirement fora separate regulatory protein, we have examined the abilityof Ca2+ to stimulate glycerophosphate dehydrogenase activity after polyacrylamide gel electrophoresis in the presence of Triton. Protein stainingof the resulting gel demonstrated resolution of several discrete bands. Glycerophosphate dehydrogenase activity staining showed a single band a t RF = 0.31 (Fig. 5). Quantitation of product formation by integration of spectrophotometerscans verifies thatthe greater than 2-fold stimulation by Ca" is preserved (Fig. 5).

C "

.

DISCUSSION

06

LA

0 6

Iloo1

lo-

phate 08060 4 -

0 2 -

FIG. 5. Ca2+ stimulation of glycerophosphate dehydrogenase activity in polyacrylamide gels. Solubilized glycerophosphate dehydrogenase (54 pg of protein/gel) was electrophoresed in 6-mm diameter gels consisting of 5% acrylamide, 0.4% bisacrylamide. and 0.18 Triton X-100.Upper. gels a and b were stained for glycerophosphate dehydrogenase activity at 1 mM glycerophosphate. Gel a. 2 mM

It is established that catecholamines cause a rise in the cytosolic Ca2' concentration of rat hepatocytes (25) and that they cause cytosolic pyridine nucleotides to become oxidized as mitochondrial NADH/NAD increases(26). Studies of gluconeogenesis from reduced substrates entering the pathway at triose phosphate (2),as well as ethanol oxidation by hepatocytes (3), indicate catecholamine stimulation of the glycerophosphate cycle. Stimulation of mitochondrial glycerophosphate dehydrogenase by Ca2', as reported here, is consistent with the hypothesis that catecholamine stimulation of reducing equivalent transfer islocalized a t this enzyme. Although a previous report (8) failed to reveal Ca2' stimulation of rat liver mitochondrial glycerophosphate dehydrogenase, this could be the resultof improper assay conditions. The concentration of electron acceptor (INT) used in that study was 1/400 of that used in our studies. I t is likely that the resulting low activity (lessthan 10% of that obtained with 0.2% INT) and the Cat+ interference in the assay precluded demonstration of a Ca2' stimulation. The sensitivity of glycerophosphate dehydrogenase toCa2' is in the range of concentrations reported for the cytosol of the hepatocyte(25). Our results indicate half-maximally effective Ca" concentrations of 0.1-1 p ~comparable , to the results of Hansford and Chappell(7) for flight muscle mitochondria. However, Fisher et al. (6) report a maximal Ca2+ stimulation at 0.1 p~ for the enzyme of rat lung mitochondria. Thiscould result from an insufficient EGTA concentration (200 p ~ to) achieve complete Ca" buffering. The K,,, (glycerophosphate)values in thepresenceand absence of Ca2' and the Ca2+ concentration required halffor maximal stimulation of the glycerophosphate dehydrogenase of hyperthyroid rat liver mitochondria are comparable to those of the enzyme from euthyroid rats. Furthermore, the chromatographic and electrophoretic behaviorof the enzyme from each source is similar (27). Carnicero etal. (lo), however, reported that theK,,, (glycerophosphate) of liver glycerophosEGTA present; gel b, 20 PM free Ca" present. Gel c was stained for protein with Coomassie blue. Lower, spectrophotometric scans of electrophoresis gels after glycerophosphate dehydrogenase activity stains. Numbers within parentheses indicate relative activitiesas determined by integrated peak areas. A, glycerophosphate omitted; B, glycerophosphate + EGTA; C , glycerophosphate + Ca".

Ca2+Stimulates Glycerophosphate Dehydrogenase phate dehydrogenase is increased in the hyperthyroid state. As previously suggested (28), the rate of glycerophosphate oxidation may have been limited by oxidative phosphorylation since the assays (10) were performed with intact mitochondria in the absence of uncoupler or phosphate acceptor. An alternate explanation may be differingconcentrations of free Ca" present during the assays of hyperthyroid versus normal mitochondria since the incubations of Carnicero et al. (10) contained no EGTA.3 The smaller aliquots of mitochondria necessary todetect glycerophosphate dehydrogenase from hyperthyroid animals wouldyield proportionately smaller amounts of endogenous calcium. For hyperthyroid mitochondria, Carnicero et al. (10) reported a K, for glycerophosphate . agrees well with the value we obtained in the of 3.4 m ~This absence of Ca2+with the INT-coupled assay, supporting the conclusion that the glycerophosphate dehydrogenase and its regulation by Ca2+are not altered during hyperthyroidism. Both WoNrab (29) and Fisher et a2. (6) reported that Ca2+ increases ADP-supported glycerophosphate respiration by isolated mitochondria even at saturating glycerophosphate concentrations. However, in the fist study, the rateof respiration by blowfly flight muscle mitochondria in the absence of Ca2+ continually diminished with time. The low rate of respiration just before Caz+ addition was taken as the rate without Ca2+.Fisher et al. (6), using lung mitochondria, demonstrated linear ratesof respiration, However, Mg2+was also able to stimulate ADP-supported respiration (6). With liver mitochondria, rates of ADP-supported glycerophosphate oxidation were line&, but CaZ+( M ) stimulation was apparent only at low and not saturating glycerophosphate concentrations (data not shown). This effect solely onthe K, is consistent with the direct assay of the enzyme reported here. Hansford and Chappell (7) solubilized glycerophosphate dehydrogenase from insect flight muscle and reported a complete loss of calcium sensitivity. Lloyd and Harrison (30) demonstrated that Ca2+stimulation of the enzyme fromflight muscle mitochondria treated with Triton X-100 was comparable to that of intact mitochondria, but Ca2+stimulation of the solubilized enzyme wasreported to be lost upon storage. We find Ca2+stimulation of the solubilized liver enzyme lost is on overnight storage at 4 "C or -20 "C unless glycerol and dithiothreitol are added to the preparation. Solubilization of glycerophosphate dehydrogenase from other sources leads to an increase in the K , for glycerophosphate (15, 30, 31). We observe a similar increase of the K , for glycerophosphate of rat liver mitochondrial glycerophosphate dehydrogenase after Triton solubilization, but the 2-fold decrease of K , by calcium ions is preserved. We found no evidence forthe involvement of calmodulin in the Ca2' stimulation of glycerophosphate dehydrogenase. The absence of an effect of added calmodulin and the slight inhibition by high concentrations of phenothiazines (Table 11) that is overcome by higher Ca2+concentrations are features that distinguish this enzyme fromothers known to be calmedulin sensitive, such as CAMP-phosphodiesterase, Ca2+-ATPase, and phosphorylase kinase (32). Moreover, the ability of CaZ' to stimulate glycerophosphate dehydrogenase in situ after electrophoresis in the presence of EDTA suggests that The 2 m~ EGTA, as present in the experiments reported here, is adequate to prevent any significant increase of free CaZ' during mitochondrial calcium release.

12771

if a Ca2+-bindingprotein is involved, it would have to be tightly associated with the enzyme. It is still possible that calmoddin is a tightly bound subunit of glycerophosphate dehydrogenase, similar to the situation with phosphorylase kinase. However, phosphorylase kinase is stimulated by additional calmodulin (33) and glycerophosphate dehydrogenase is not. The simplest interpretation is that Ca2+binds directly to glycerophosphate dehydrogenase to activate it. Since the enzyme is located on the cytosolic faceof the inner mitochondrial membrane and responds to Ca2' at prevailing cytosolic concentrations, and since catecholamines raise cytosolic Ca2+ levels, we suggest glycerophosphate dehydrogenase as a site of catecholamine action in the rathepatocyte. REFERENCES 1. Dawson, A. G . (1979) Trends Biochem. Sci. 4,171-176 2. Kneer, N.M., Wagner, M. J., and Lardy, H. A. (1979) J. Biol. Chem. 254,12160-12168 3. Ochs, R. S., and Lardy, H. A. (1981) FEBS Lett. 131, 119-121 4. Werner, H.V., and Berry, M. N. (1974) Eur. J.Biochem. 42,315324 5. Khgenberg, M. (1970) Eur. J. Biochem. 13,247-252 6. Fisher, A. B., Scarpa, A., LaNoue, K. F., Bassett, D., and Williamson, J. R. (1973) Biochemistry 12, 1438-1445 7. Hansford, R. G., and Chappell, J. B. (1967) Biochem. Biophys. Res. Conzmun. 27,686-692 8. Zammit, V. A., and Newsholme, E. A. (1976) Biochem. J. 154, 677-687 9. Lee, Y. P., and Lardy, H. A. (1965) J. Biol. Chem. 240,1427-1436 10. Carnicero, H. H., Moore, C.L., and Hoberman, H. D. (1972) J. Biol. Chem. 247,418-426 11. Ochs, R. S., Wernette, M. E., and Lardy, H.A. (1980) Fed. Proc. 39, 1928 12. Wernette, M. E. (1981) Fed. Proc. 40, 1886 13. Johnson, D., and Lardy, H. A. (1967) Methods Enzymol. 10,9496 14. Schnaitman, C., and Greenawalt, J . W. (1968) J. Cell Biol. 38, 158-175 15. Cole, E. S., Lepp, C. A., Holohan, P. D., and Fondy, T. P. (1978) J. Biol. Chem. 253,7952-7959 16. Layne, E. (1957) Methods Enzymol. 3,447-454 17. Bradford, M. (1976) Anal. Biochem. 72, 248-254 18. Gardner, R. S. (1974) Anal. Biochem. 59,272-276 19. Portzehl, H., Caldwell, P. C., and Riiegg, J. C. (1964) Biochim. Biophys. Acta 79,581-591 20. SiUen,L. G., and Martell, A. E. (1964) Stability Constants of Metal-Ion Complexes, Chemical Society, London 21. Pennington, R. J. (1961) Biochem. J. 80,649-654 22. Dewald, B., Duianey, J. T., andTouster, 0. (1974) Methods Enzymoi. 32, Part B, 82-91 23. NacNas, M. M., Margulies, S. I., and Seligman, A. M. (1960) J. Biol, Chem. 235,499-503 24. Levin, R. M., and Weiss, B. (1976) Mol. Pharmacoi. 12, 581-589 25. Murphy, E., Coll, K., Rich, T. L., and Williamson, J. R. (1980) J. Biol. Chem. 255,6600-6608 26. Yip, B., and Lardy, H.A. (1981) Arch. Biochem. Biophys., in press 27. Chen, C. (1979) Ph.D. thesis, University of North Dakota 28. Hensgens, L. A. M., Nieuwenhuis, B. J. W. M., VanderMeer, R., and Meijer, A. J. (1980) Eur. J. Biochem. 108,39-45 29. Wohlrab, H. (1977) Biochim. Biophys. Acta 462, 102-112 30. Lloyd, W. J., and Harrison, R. (1974) Arch. Biochem. Biophys. 163,185-190 31. Cottingham, I. R., and Ragan, C. I. (1978)Biochem. Soc. Transns. 6, 1307-1310 32. Cheung, W.Y. (1980) Science 207, 19-27 33. Shenolikar, S., Cohen, P. T. W., Cohen, P., Nairn, A. C., and Perry, S. V. (19791 Eur. J. Biochem. 100,329-337