Studies on adenosine triphosphate transphosphorylases. XVII. A physicochemical comparison of the ATP-creatine transphosphorylase (creatine kinase) isozymes from man,calf, and rabbit

All of the creatine kinase isozymes from human, calf, and rabbit brain and muscle are composed of two noncovalently linked polypeptide chains, based upon sedimentation equilibrium analyses in the presence and absence of disruptive agents. The brain-type isozymes of man, calf, and rabbit proved to be slightly heavier than the muscle types. Various physicochemical properties of the isozymes are recorded. Each group of isozymes, i.e., the muscle, hybrid (muscle-brain), and brain isozymes from man, calf, and rabbit, showed similar electrophoretic behavior, although isoelectric points were not precisely identical for the muscle and hybrid types. Theoretical titration curves constructed from amino acid compositions of the calf isozymes showed reasonable agreement between their calculated and measuredpI ₀ values (isoelectric point extrapolated to zero ionic strength). The three native muscle isozymes and brain isozymes all contain two reactive sulfhydryl groups per mole or one per polypeptide chain of their two-chain proteins, which may be titrated with 5,5′-dithiobis (2-nitrobenzoic acid); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of ten- SH groups per mole of each brain-type and eight- SH groups per mole of muscle-type isozyme in the case of man, calf, and rabbit. A comparison of their amino acid compositions and tryptic peptide maps shows that there is only a slightly greater degree of homology between the individual isozymes of the same type (muscle type or brain type) than between the muscle- and brain-type isozymes of the same species.     

Cytoplasmic and Mitochondrial Creatine Kinases from the Skeletal Muscle of Sperm Whale (Physeter macrocephalus). Molecular Cloning and Enzyme Characterization

We have amplified two cDNAs, coding for creatine kinases (CKs), from the skeletal muscle of sperm whale Physeter macrocephalus by PCR, and cloned these cDNAs into pMAL plasmid. These are the first CK cDNA and deduced amino acid sequences from cetaceans to be reported. One of the two amino acid sequences is a cytoplasmic, muscle-type isoform (MCK), while the other was identified as a sarcomeric, mitochondrial isoform (sMiCK) that included a mitochondrial targeting peptide. The amino acid sequences of sperm whale MCK and sMiCK showed 94–96% sequence identity with corresponding isoforms of mammalian CKs, and all of the key residues necessary for CK function were conserved. The phylogenetic analyses of vertebrate CKs with three independent methods (neighbor-joining, maximum-likelihood and Bayes) supported the clustering of sperm whale MCK with Bos and Sus MCKs, in agreement with the contemporary view that these groups are closely related. Sperm whale MCK and sMiCK were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and the kinetic constants (K _m, K _d and k _cat) were determined for the forward reaction. Comparison of kinetic constants with those of human and mouse CKs indicated that sperm whale MCK has a comparable affinity for creatine (K _m^Cr = 9.38 mM) to that of human MCK, and the sMiCK has two times higher affinity for creatine than the human enzyme. Both the MCK and sMiCK of sperm whale display a synergistic substrate binding (K _d /K _m = 3.1–7.8) like those of other mammalian CKs.     

Comparisons of creatine kinase primary structures

Comparisons of nine creatine kinase sequences show that 67% of the protein sequence is identical among rabbit, rat, mouse, and chicken muscle, rabbit, rat, and chicken brain, and electric organ sequences from two species of electric ray(Torpedo). The extensive homology precludes a facile prediction of active-site residues based on sequence conservation. The sequences are more similar within isozyme types than are the different isozymes from any one species. There are 35 positions in the muscle and brain sequence pairs for three species which differentiate the two forms. TheTorpedo sequences do not fall completely into either of these patterns. Except for homology with partial sequences of other ATP-guanidino phosphotransferases, no significant homology with other protein or nucleic acid sequences in available databases was found. Preliminary secondary structural predictions suggest that the C-terminal half of the protein is likely an /-type protein. Placement in the sequence of two peptides found in previous cross-linking studies reveals two stretches of primary structure that are presumably close in space to the reactive Cys-283 and hence close to the active site.     

Studies on muscular dystrophy: a comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of Duchenne muscular dystrophy.

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem.252, 8382–8390) have now been compared physically and chemically with their normal human counterparts (viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s_20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type (viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs₂ and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k_{second order} values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs₂, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2-¹⁴C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2-¹⁴C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH₂COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH₂COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.     

Uptake of creatine by cultured cells

We have examined the uptake of creatine by cultured monolayers of human IMR-90 flbroblasts, human uterine smooth muscle cells, calf aortic smooth muscle cells, and myoblasts and myotubes of the L₆E₉ rat skeletal muscle cell line. Creatine uptake is dependent on temperature and sensitive to the presence of Na⁺ in the extracellular medium. It is saturable, apparently concentrative, and inhibited by ouabain and structural analogs of creatine. In these respects, it resembles the process of creatine uptake by isolated preparations of skeletal muscle and brain tissues. Lineweaver-Burk plots of the data for variation in rate of uptake with concentration of creatine in the medium are nonlinear, suggesting that the process of uptake may be heterogeneous. Assuming the operation of two saturable processes of uptake, we calculated two values for apparent K_m and V for each cell line. Kinetic parameters of creatine uptake by the different cell types are similar. The lower values of K_m (0.02–0.04 mm) are in the physiological range of creatine concentration in mammalian plasma.     

Interaction of Creatine Kinase from Monkey Brain with Substrate: Analysis of Kinetics and Fluorescence Polarization

Abstract: Titrimetric determination of the dissociation constants for the binding of substrates to creatine kinase from monkey brain reveals 13-fold and 4-fold synergism in the forward and reverse directions, respectively. This synergism is expressed as a decrease in the K_D for a given substrate in the ternary complex compared with the binary complex and may be a reflection of substrate-induced conformational change. Creatine kinase labeled with two molecules of 5′-iodoacetamidofluorescein displays a blue shift and a decrease in fluorescence intensity upon binding of MgADP, indicative of movement of the dye into a more hydrophobic environment and quenching of the extrinsic fluorescense. Rotational relaxation times determined from analysis of fluorescence polarization of dansylated brain creatine kinase decrease from 212 ± 7 ns to 189 ± 6 ns upon MgADP binding. Dansylated creatine kinase in 0.5% sodium dodecyl sulfate has a rotational relaxation time of 135 ± 6 ns. The rotational relaxation time of dansylated muscle-type isoenzyme is unaffected by MgADP and has the same value as the brain isoenzyme-MgADP complex. Polarization values at 25°C for muscle and brain enzyme labeled with 3 - (4 - maleimidylphenyl) - 7 - diethylamino - 4 - methylcoumarin compared with limiting polarization and polarization of the free dye suggest that the dye rotation is severely restricted in the muscle form, but possesses freedom of rotation in the brain form. These results support the conclusion that compared with the muscle isoenzyme, the brain isoenzyme is more open at the active site and more flexible overall. Binding of MgADP by brain creatine kinase produces a protein more compact across one or both of its rotational axes, thus resembling the conformation of the muscle isoenzyme. It is probable that creatine kinase in the brain, unlike that from muscle, is subject to kinetic regulation accompanied by conformational modification. This suggests that the neurobiochemical role of the brain isoenzyme is distinct from the metabolic function of the muscle isoenzyme.     

Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.     

A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the α-class isozymes of GST. It is characterized by kinetic properties intermediate between the α- and π-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P. et al. (1992) FEBS Lett., 313, 173–176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other α-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M_r values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxidase suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.     

Purification and cell-free translation of a unique high molecular weight form of the brain isozyme of creatine phosphokinase from mouse

Mouse brain creatine kinase was purified to homogeneity and shown to consist of two polypeptide chains of 50,000 daltons. This protein thus differs in size from all other creatine kinase molecules purified to data including the mouse muscle enzyme which shows a molecular weight between 39,000 and 42,000. The high molecular weight isozyme has been shown to represent the primary translation product of creatine phosphokinase mRNA from mouse brain. The unusual size of this creatine phosphokinase subunit provides unique tools for the study of the differential regulation of creatine kinase gene expression and for the study of subunit interactions in creatine kinase isozymes.     

Structural relatedness of mouse lactate dehydrogenase isozymes,A4 (muscle), B4 (heart), and C4 (testis)

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A₄), LDH-H(B₄), and LDH-X(C₄), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Concanavalin A-Sepharose Chromatography of Hexosaminidase Isozymes Secreted By Tetrahymena*

Tetrahymena pyriformis strain HSM secretes 4 isozymes of hexosaminidase. Purified isozymes B₁ and B₂ are eluted from the void volume of a concanavalin A-Sepharose column, suggesting that they are not glycosylated. Purified isozymes A₁ and A₂ bind to the column and are eluted at ～0.1 M α-methylmannoside, suggesting that these isozymes are glycoproteins. In agreement with earlier deductions based on a differential kinetic assay for the A and B isozymes, the elution pattern of hexosaminidase activity from material secreted by cells grown to early and late stationary phase was consistent with these secretions containing primarily the B and the A isozymes, respectively.     

Genetic and molecular analysis of nonrandom dimer assembly of the creatine kinase isozymes of fishes

Species within many families of actinopterygian bony fishes (class Osteichthyes) have a two-banded allelic isozyme phenotype in individuals heterozygous at the creatine kinase A locus. This two-banded pattern is formed by the presence of the two homodimeric isozymes and the absence of the expected heterodimer. Sharks and amphibians have retained the ability to form all three allelic isozymes in individuals which are heterozygous. Reversible denaturation procedures were able to assemble the different allelic CK-A subunits within a species to form CK-A₂ heterodimers. Furthermore, heterodimers were formed from different CK-A subunits from highly divergent species after this in vitro molecular hybridization process. It is concluded from these studies that the polypeptidebinding sites of creatine kinase are structurally conservative in most fishes and that the absence of a heterodimer in heterozygous individuals is not due to a structural incompatibility between the different A subunit types or to an instability of the heterodimer during electrophoresis. A temporal and/or spatial isolation of allelic CK-A subunit synthesis and assembly, within differentiated skeletal muscle, appears to have evolved in the actinopterygian bony fishes.This research was supported by NSF Grant PCM76-08383 to G. S. W. and by a NIH Cell and Molecular Biology Traineeship to S. D. F.     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K₂L₂ and K₃M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K₄ also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M₄. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.     

Kinetic evidence for human liver and stomach aldehyde dehydrogenase-3 representing an unique class of isozymes

Substrate and coenzyme specificities of human liver and stomach aldehyde dehydrogenase (ALDH) isozymes were compared by staining with various aldehydes including propionaldehyde, heptaldehyde, decaldehyde, 2-furaldehyde, succinic semialdehyde, and glutamic -semialdehyde and with NAD⁺ or NADP⁺ on agarose isoelectric focusing gels. ALDH3 isozyme was isolated from a liver via carboxymethyl-Sephadex and blue Sepharose chromatographies and its kinetic constants for various substrates and coenzymes were determined. Consistent with the previously proposed genetic model for human ALDH3 isozymes (Yinet al., Biochem. Genet. 26:343, 1988), a single liver form and multiple stomach forms exhibited similar kinetic properties, which were strikingly distinct from those of ALDH1, ALDH2, and ALDH4 (glutamic -semialdehyde dehydrogenase). A set of activity assays using various substrates, coenzymes, and an inhibitor to distinguish ALDH1, ALDH2, ALDH3, and ALDH4 is presented. As previously reported in ALDH1 and ALDH2, a higher catalytic efficiency (V _max/K _m) for oxidation of long-chain aliphatic aldehydes was found in ALDH3, suggesting that these enzymes have a hydrophobic barrel-shape substrate binding pocket. Since theK _m value for acetaldehyde for liver ALDH3, 83 mM, is very much higher than those of ALDH1 and ALDH2, ALDH3 thus represents an unique class of human ALDH isozymes and it appears not to be involved in ethanol metabolism.This work was supported by grants from the National Science Council and the Academia Sinica, Republic of China.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

Studies on phosphorylase isozymes in lower vertebrates: evidence for the presence of two isozymes in elasmobranchs.

Two distinct phosphorylase isozymes, skeletal muscle phosphorylase b and liver phosphorylase b, have been purified from skate (Raja pulchra) in a homogeneous form as judged by electrophoretic and immunological criteria. Both isozymes were dependent on AMP for activity and converted to a forms by rabbit muscle phosphorylase kinase. Their subunit molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was 94,000. These isozymes were distinctly different in affinities for glycogen and AMP, while they were very similar in sensitivities to SO₄^2?. Rabbit antibodies against each of the muscle and liver isozymes inhibited completely the respective specific antigens. No cross-reaction was observed in double diffusion tests, but some immunological relatedness of these isozymes was demonstrated by inhibition tests with antibodies. Their similarity was also shown by amino acid analyses. No evidence has been obtained that the skate possesses such an isozyme as mammalian phosphorylase L, the b form of which is inactive even in the presence of AMP. Electrophoretic studies on phosphorylases of crucian carp, toad, and snake revealed that these animals possess three isozymes which strikingly resemble mammalian isozymes in the organ-specific distribution and electrophoretic behavior.     

Purification and characterization of cytoplasmic creatine kinase isozymes ofXenopus laevis

The soluble creatine kinase isozymes CK-II, CK-III, and CK-IV fromXenopus laevis have been purified to apparent homogeneity and their subunits characterized by means of molecular weight, peptide pattern, and dissociation-reassociation experiments. CK-III and CK-IV are homodimeric isozymes whose subunits are distinct in both molecular weight (42,000 and 41,000, respectively) andStaphylococcus aureus V₈ peptide pattern. In dissociation-reassociation experiments, those two subunits do form active heterodimeric isozymes with one another or with rabbit M-CK subunits. Hybrid CK-III/IV isozymes occur also during embryonic differentiation and in adult heart muscle, whereas most other adult tissues contain only homodimeric CK-III or CK-IV isozymes. The CK-II isozyme is a heterodimer composed of one CK-III subunit and another subunit specific to CK-II (M _r =41,000). Neitherin vivo norin vitro does this subunit seem able to form homodimers or heterodimers with CK-IV and rabbit M-CK subunits. If we take into account the apparent association of CK-I isozyme with cellular organelles, these results corroborate earlier statements and suggest that the CK isozyme system ofX. laevis is encoded by at least four differentially regulated genomic loci.