Squalus acanthias and Raja tengu. BY. A. HASNAIN and T. YASUI. (Department of Animal Science, Faculty of Agriculture, Hokkaido University, Sapporo, Japan).
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Archives Internationales de Physiologie et de Biochimie, 1986, 94, 233-231
Recu le 5 fevrier 1986.
Urea tolerance of myofibrillar proteins of t w o elasrnobranchs : Squalus acanthias and Raja tengu BY
A. HASNAIN and T. YASUI (Department of Animal Science, Faculty of Agriculture, Hokkaido University, Sapporo, Japan)
(2 figures)
Some biochemical properties of actomyosin and myosin from elasmobranchs, Squalus acanthias and Raja tengu are compared with those of a freshwater (Cyprinus carpio) and a marine teleost (Seriola quinquiradiata). Whereas Ca2+-ATPaseof teleost actomyosins are more stable in the absence of urea, the reverse is true for elasmobranchs up to 1.0 M urea. In contrast to that of teleosts, the Mg2+-ATPase of S. acanthias actomyosin shows an activation in the presence of urea, where as that of R. tengu persists. Below 1 .O M urea, there is low incorporation of DTNB into thiols of elasmobranch myosins, and losses in a-helicity are reversible up to 5.0 M urea. The results, thus, demonstrate that for a certain concentration of urea, elasmobranch myofibrillar proteins may exhibit a group specific tolerance to urea.
Introduction
It has been found that enzymes of poikilotherms are better catalysts at lower temperatures than those of homotherms (Low et al., 1973). This catalytic efficiency has probably been achieved at the expense of the relative stability (HOCHACHKA & SOMERO,1973). Whereas the temperature appears the most dominant factor in most of the cases, there may be some other physico-chemical factors also which have contributed to the final fitness of various enzymes in terms of structure and function. An interesting example is high blood urea level in some organisms resulting in a & SOMERO,1973). These switchover from aminotelism to ureotelism (HOCHACHKA organisms include some amphibians and elasmobranchs which may maintain a urea & BROWN,1961). Data dealing concentration as high as 0.75 M in blood (PROSSER with urea adaptability are scarce and somewhat contradictory. For instance, MALWSZ (1974) and MALYUSZ & THIEMAN (1976) found no specific adaptation to urea of some et al. (1974) sarcoplasmic enzymes of dogfish. On the contrary, BONAVENTURA reported functional tolerance in elasmobranch haemoglobins to urea level as high as 5.0 M.
Abbreviation used : ORD, optical rotatory dispersion.
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A. HASNAIN AND T. YASUI
In this report we present comparative data to demonstrate urea adaptability of myofibrillar proteins of two elasmobranchs.
Materials and Methods Ice-stored, fresh or frozen samples of Squalus acanthias (dogfish), Raja tengu (ray) and Seriola quiquiradiata (yellowtail) were used. Frozen or fresh samples of muscle from carp (Cyprinus carpio) were utilized. Actomyosin and myosins were prepared according to established procedures (ARAI et al., 1976; HASNAIN et al., 1979). Preparations showing the degradation of myosin heavy chain on SDS polyacrylamide gels, which was performed according to WEBER& OSBORN (1969) were rejected. ATP sensitivity of myosin and actomyosin preparations were respectively 5% and 80%. It was measured with Ostwald viscometers at 20°C using the equation : [(log v,-log r]&p)log r]ATp] x 100, where r],, and vATpare viscosities relative to protein solvent with or without ATP. All urea solutions contained 0.5 M or 0.6 M KCI and 20 mM Tris-HC1 buffer pH 7.5, which were the actual solvents for myosin or actomyosin. To obtain a fixed final molarity, one volume of protein solution (3-10 mg/ml) was mixed with an equal volume of solution at twice the wanted urea concentration. Ca2'-ATPase was assayed in 50 or 60 mM KCI, 25 mM Tris-maleate buffer pH 7.0, 5 mM CaCl, and 1 mM ATP at 20°C. CaC1, was replaced by MgCI, to assay actomyosin Mg2'-ATPase, and the temperature of assay was varied. Details of thermal and urea inactivation have been published elsewhere (ARAIet al., 1976). ORD spectra were recorded with a Jasco ORD/UV spectropolarimeter and the helicity was calculated according to standard formula as quoted previously (HASNAIN et al., 1979). Difference spectra were measured on a Hitachi model 323 automatic temperature controlled spectrophotometer. DTNB incorporation experiments were performed as described previously (HASNAIN et al., 1979).
Results Myosins from dogfish and yellowtail were highly thermolabile, therefore their actomyosins were used for the inactivation experiments. It is however, well established that the relative interspecies order of stability is not affected by the presence of actin
Helical contents (Vo) Species Original
Dogfish Ray Carp Yellowtail
56.0 62.8 58.1
61.6
1.0 M urea
After dialysis
49.1 59.4 44.0 50.1
56.0 62.4 51.6 54.1
5.0 M
After dialysis
urea 12.6 19.8 21.8 18.9
53.5 62.4 39.9 41.5
All samples were incubated overnight with urea at a temperature of 4°C prior to ORD measurements.
235
UREA TOLERANCE OF MYOFIBRILLAR PROTEINS OF ELASMOBRANCHS
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FIG.1.
25
20 15 10 5 Temperature (“CI
1A & B, Arrheniusplots showing the temperature dependence of velocity constants (kd)of inactivation of actomyosin Ca”-A TPase in the absence and the presence of 1.0 M urea, respectively. C & D,Physiological (Mg”) A TPase activities of teleost and elasmobranch actomyosins. respectively. Continuous curves are the relative values in the presence of 0.2 M and discontinuous ones in the ; ray, 4-; carp, -0-and yellowtail, -.-). The relative presence of 0.8 M urea (dogfisch value has been expressed as the percentage of the highest value obtained in the absence of urea at a particular temperature. The arrow indicates the total loss of activity.
*-
which plays a stabilizing rdle without affecting the kinetics (HASNAINet al., 1973; ARAIet al., 1976). In the absence of urea dogfish- and ray actomyosin Ca”-ATPases were the least stable (Fig. 1A) whereas the presence of the denaturing agent reversed this order and the teleost ATPases showed the highest instability (Fig. 1B). This is in contrast with the general trend observed for mammalian and teleost actomyosins and myosin ATPet al., 1973), where the addition of urea increased thermal instabilities ase (HASNAIN in the same order. In the presence of up to 0.8 M urea dogfish actomyosin Mg2+-ATPaseactivity was remarkably activated in the physiological temperature range (Fig. 1C). Although some decrease was observed in ray actomyosin ATPase, its behaviour was unlike those of the teleosts; since the activity persisted even at the low temperatures. The temperatures where teleost actomyosins exhibit some activity are above the expected body temperatures which in poikilotherms should correspond the temperature of the habitat ( < 15OC).
236
A. HASNAIN AND T. YASUI
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FIG. 2 A : The dependence of DTNB incorporation into thiols of various myosins. Symbols are the sance as in Fig. I . Typically, 2 ml solutions containing 2 mg/ml protein were mixed with equal amount of urea solutions of fixed molarity directly in photometric cuvette followed by addition of 20 11 DTNB solution. All additions were made within 10 s and incubations done at 5°C. B & C : Typical difference of carp (B)and dogfish ( C ) myosin in urea solutions. The spectra of ray and yellowtail were similar to respective teleost and elasmobranch myosins. The spectra were recorded within 10 min following addition of urea. The final urea concentrations were: 0.5 M ( - * - ) ; 1.0 M ( - - - ) a n d 5.0 M (++-).
UREA TOLERANCE OF MYOFIBRILLAR PROTEINS OF ELASMOBRANCHS
237
Whereas the reversibility of urea-induced losses in a-helicity is higher in elasmobranch myosins (Table I), the rate of incorporation of DTNB into thiols in the presence of physiological concentrations of urea (
