Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase

The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the adenylate kinase isozymes from the serum and erythrocyte of normal and Duchenne dystrophic patients. Isolation, physicochemical properties, and several comparisons with the Duchenne dystrophic aberrant enzyme

Two species of adenylate kinase isozymes (ATP:AMP phosphotransferase, EC 2.7.4.3) from human Duchenne dystrophic serum were separated by Blue Sepharose CL-6B affinity column chromatography. One of these species was the "aberrant" adenylate kinase isozyme, found specifically in the Duchenne type of this disease (Hamada, M., Okuda, H., Oka, K., Watanabe, T., Ueda, K., Nojima, M., Kuby, S.A., Manship, M., Tyler, F., and Ziter, F. (1981) Biochim. Biophys. Acta 660, 227-237). The separated aberrant form possessed a molecular size of 98,000 (+/- 1,500), whereas the normal serum species of the enzyme was 87,000 (+/- 1,600) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by gel filtration, and by sedimentation equilibrium. The sedimentation coefficient of each species was found to be 5.8 S for the aberrant form and 5.6 S for the normal form, respectively. The subunit size (Mr = 24,700) of the aberrant enzyme in 8 M urea proved to be very similar to that of the normal human liver enzyme (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S.A. (1982) J. Biol. Chem. 257, 13120-13128), and the normal species subunit (Mr = 21,700) was found to be very similar to that of the normal human muscle enzyme (Kuby, S.A., Fleming, G., Frischat, A., Cress, M.C., and Hamada, M. (1983) J. Biol. Chem. 258, 1901-1907). Both species were tetrameric enzymes in the serum. The amino acid composition for the normal species was similar to that for the muscle-type enzyme, and that for the aberrant species was similar to the liver enzyme, but with some notable exceptions in both cases. Thus, the normal species had no tryptophan and two half-cystine residues/subunit; whereas, there was 1 tryptophan and 4 half-cystine residues/subunit of the aberrant molecule. The amino acid composition of both serum isozymes when compared to their respective muscle or liver-type enzyme differed mainly in the content of Glu, Asp, His, Leu, Ile, Gly. Kinetic properties of the two forms of human serum adenylate kinase were studied at limiting concentrations of both ADP3- and MgADP- in the reverse reaction and of AMP2- and MgATP2- in the forward reaction. The type of reaction mechanism compatible with the data was a two-substrate random quasiequilibrium type of mechanism without independent binding of the substrates and with a rate-limiting step largely at the interconversion of the ternary complexes.     

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.     

Inactivation of rabbit, pig, and carp adenylate kinases by N6-o- and p-fluorobenzoyladenosine 5'-triphosphates.

N6-O- and p-fluorobenzoyladenosine 5'-triphosphates (IIIc and IIc, respectively) have been synthesized as potential adenosine 5'-triphosphate (ATP) site-directed reagents for enzymes. IIc and IIIc were substrates of yeast hexokinase; neither they nor the corresponding ADP derivatives inactivated yeast hexokinase or rabbit pyruvate kinase. IIc rapidly inactivated rabbit and carp muscle adenylate kinases; the effect is probably ATP site directed because N6-benzoyl-ATP did not inactivate and was a substrate (Vmax = 28 and 10%, respectively, that of ATP), and because of ATP retarded the inactivation. The inactivations followed pseudo-firsr-order kinetics; in the presence of 2.64 mM ATP at 0 degrees the half-life of the rabbit kinase was 210 min with 50 muM IIc and the half-life of the carp kinase was 130 min with 100 muM IIc. Adenylate kinase of pig muscle was inactivated by IIc in a manner similar to the rabbit and carp enzymes except that the rate of inactivation exhibited an inflexion. IIIc inactivated rabbit, pig, and carp adenylate kinases by pseudo-first-order kinetics; the rate constants for inactivation at 0 degrees were 9.1 X 10(-3), 1.3 X 10(-3), and 1.9 X 10(-3) min-1 and the apparent dissociation constants (K) of the IIIc-enzyme complexes were 710, 970, and 720 muM, respectively. From the substrate properties of IIIc alone and in admixture with ATP its dissociation constants (Ki) from the ATP sites of the enzymes were found to be 500, 700, and 845 muM, respectively. The similarity between the K and Ki values, together with marked retardation of the inactivations by ATP, indicates that IIIc is an ATP-site-directed reagent for the three adenylate kinases.     

Characterization of calf-ovary adenosine;3':5'-monophosphate-dependent protein kinases and adenosine-3':5'-monophosphate-binding proteins.

Protein phosphokinase activity from the calf ovary cytosol (105000 X g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major protein kinases, PK-H1 and PK-H2, both dependent on adenosine 3':5'-monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic-AMP-dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK-H1 of a unique cyclic-AMP-binding protein which has little catalytic activity associated with it. The cyclic-AMP-binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit. The ovarian cyclic-AMP-dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat-stable protein inhibitor isolated from rabbit skeletal muscle. Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic-AMP-dependent protein kinases and cyclic-AMP-binding proteins which include protein kinases and binding proteins of greater than 400 000 molecular weight.     

Protein kinases of rabbit and human erythrocyte membranes. Solubilization and characterization.

Two protein kinases (EC 2.7.1.37) from rabbit and one from human erythrocyte membranes have been solubilized with 0.5 M NaCl. These enzymes have been partially purified by (NH4)2SO4 fractionation and gel filtration. The rabbit membrane enzymes have apparent Mr values of 100 000 and 30 000, as determined in the presence of 0.4 M NaCl. In the absence of salt, these enzymes aggregate into high molecular weight species. The kinase from human erythrocyte membranes has an apparent Mr of 30 000 and appears to have properties similar to those of the 30 000-dalton rabbit kinase. All three enzymes catalyze the phosphorylation of casein and phosvitin in salt-stimulated reactions. None of these enzymes appears to be related to cyclic AMP-dependent protein kinases.     

Purification and properties of rabbit liver cathepsin M and cathepsin B

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.     

Three major forms of adenylate kinase from adult and fetal rat tissues.

The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.     

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.     

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Phosphorylation of glycogen synthase and of the beta subunit of eukaryotic initiation factor two by a common protein kinase

A protein kinase from rabbit reticulocytes, able to phosphorylate the beta subunit of eukaryotic initiation factor 2 (eIF-2), has been demonstrated to phosphorylate also glycogen synthase. A glycogen synthase kinase (PC0.7) from rabbit skeletal muscle has been shown to phosphorylate the beta subunit of eIF-2. Comparison of highly purified preparations of the two protein kinases has indicated several similarities of properties. 1) Both enzymes were associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000), and exhibited apparent native molecular weights of 176,000-180,000 by gel filtration and 130,000-140,000 by sucrose density gradient sedimentation. 2) Both enzymes phosphorylated glycogen synthase, eIF-2 beta, phosvitin, and casein and were effective in utilizing GTP and ATP as phosphoryl donors. 3) Both enzymes displayed the same chromatographic behavior on phosvitin-Sepharose, phosphocellulose, and DEAE-cellulose. 4) Both enzymes underwent an autophosphorylation of the beta polypeptide when incubated with ATP and Mg2+. On the basis of these and other properties, we propose that the two protein kinases, if not identical, are very similar enzymes.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

Evidence for the identity of nuclear and cytoplasmic adenosine-3':5'-monophosphate-dependent protein kinase from porcine ovaries and nuclear translocation of the cytoplasmic enzyme.

Protein phosphokinase activity from a 0.5 M NaCl extract of purified porcine ovary nuclei has been resolved by Sephadex G-200 gel filtration into three forms of kinase, protein kinase I and III, both independent of adenosine 3':5'-monophosphate (cyclic AMP), and cyclic-AMP-dependent protein kinase II. Cyclic AMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II, and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria [Traugh, J.A., Ashby, C.D. and Walsh, D.A. (1974) nuclear protein kinase II can be classified as cyclic-AMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cyclic-AMP-independent enzymes. Evidence for the identity of nuclear cyclic-AMP-dependent protein kinase II with cytosol (105 000 X g supernatant fraction) cyclic-AMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cyclic-AMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 muM, showed similar substrate specificity, and revealed similar antigenic properties. Cyclic-AMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cyclic-AMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cyclic-AMP-dependent protein kinase with 0.1 muM 8-p-chlorophenylthio cyclic AMP, considerable cyclic-AMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. From these data it is concluded that the nuclear cyclic-AMP-dependent protein kinase II is related to or identical with the ovary cytoplasmic cyclic-AMP-dependent protein kinase, supporting the concept that nuclear cyclic-AMP-dependent protein kinase is of cytoplasmic origin.     

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Primary and tertiary structure of the principal human adenylate kinase.

1. Human adenylate kinase (isoenzyme AK-1-1) from skeletal muscle is a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine at the C-terminus. 2. The primary structure of the enzyme was determined: Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Thr-Lys-Ile-Ile-Phe-Val-Val-Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly-Asp-Leu-Leu-Arg-Ser-Glu-Val-Ser-Ser-Gly-Ser-Ala-Arg-Gly-Lys-Lys-Leu-Ser-Glu-Ile-Met-Glu-Lys-Gly-Gln-Leu-Val-Pro-Leu-Glu-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met-Val-Ala-Lys-Val-Asn-Thr-Ser-Lys-Gly-Phe-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Gln-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Arg-Ile-Gly-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp-Ala-Gly-Pro-Glu-Thr-Met-Thr-Arg-Arg-Leu-Leu-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp-Asn-Glu-Glu-Thr-Ile-Lys-Lys-Arg-Leu-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val-Arg-Lys-Val-Asn-Ala-Glu-Gly-Ser-Val-Asp-Glu-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu-Asp-Ala-Leu-Lys. 3. When the primary structure of the human enzyme was fitted to the electron density map of porcine adenylate kinase, all nine amino acids which are different in the homologous enzymes from pig and man were located on the surface of the molecule. 4. Precession photographs of crystalline human and of crystalline porcine adenylate kinase corroborated the result that the polypeptide chains of the two enzymes are folded in a closely related manner. 5. The structure of human adenylate kinase incorporates the so-called nucleotide-binding domain which is present in a wide variety of proteins in nature. Some implications of this phenomenom for the molecular biology and the molecular pharmacology of man are discussed.