Sequestration of adenosine in crude extract from mouse liver and other tissues.

Adenosine (1 microM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Saeb?, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333--340). These data point to a role of this protein in the sequestration of adenosine in crude extract. The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed. The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 microM. The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract. The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver. A fraction of adenosine bound is converted to adenine.

1. Adenosine bound to the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver was partly converted to a product which was identified as adenine in four chromatographic systems. Ribose was formed in equivalent amounts. 2. The time course of the reaction was characterized by an initial burst phase lasting for less than one second followed by a slow progressive phase. The reaction was partly reversed by prolonged incubation, slow denaturation of the protein, dilution of the incubation mixture and removal of adenosine by converting it to inosine by the enzyme adenosine deaminase. 3. Both the ATP-treated (Ueland, P.M. and D?skeland, S.O. (1978) Arch. Biochem. Biophys. 185, 195--203) and the non-treated protein were subjected to polyacrylamide gel electrophoresis at pH 8.8. The adenosine-adenine, the cyclic AMP binding activities and the conversion activity comigrated with the main protein band, indicating that these properties reside on the same protein molecule. 4. Adenine generated by hydrolysis of adenosine was mainly bound to the protein as judged by nearly complete reversion of the conversion upon dilution in the presence of excess unlabelled adenine and by Sephadex G-25 chromatography. 5. The conversion of adenosine to inosine by the enzyme adenosine deaminase was decreased in the presence of the binding protein. 6. Adenine formation could also be demonstrated under condition of enzymic formation of S-adenosylhomocysteine, i.e. in the presence of hymocysteine.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver: some physicochemical properties.

A number of physiochemical properties of the cyclic AMP-adenosine binding protein of mouse liver (Ueland, P.M. and D?skeland, S.O. (1977) J. Biol. Chem. 252, 677--686) have been studied. 1. The specific extinction coefficient, E1%280nm, was estimated to 13.0. 2. Amino acid and amide group analyses confirmed the acidic properties of the protein as determined by electrofocusing (pI = 5.7). Based on the estimated partial specific volume (v = 0.74 cm3/g) the minimum molecular weight of the native, tetrameric protein was recalculated to be 185 000 (s20,w = 8.8 . 10(-13) s and Stokes radius = 48 A). 3. No NH2-terminal amino acid was found by the dansyl method using [14C]-dansyl chloride, indicating that the NH2-terminal groups are blocked. 4. Amino acid analyses gave 6 half-cystine residues per subunit, and the same number of free sulfhydryl groups was found by titration of the denatured protein with 5,5'-dithiobis (2-nitrobenzoic acid). 5. The reactivity of the SH groups in the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) revealed rapidly reacting (SHI), sluggishly reacting (SHII) and "masked" (SHIII) SH groups. ATP, adenosine, Mg2+ and KCl, factors known to affect the activation of cyclic AMP binding sites (Ueland, P.M. and D?skeland, S.O. (1978) Arch. Biochem. Biophys., in press) changed the reactivity of separate SH groups.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Distribution of adenosine deaminase-complexing protein in murine tissues

It has been suggested that mouse and rat lack adenosine deaminase-complexing protein because in these species exclusively the small molecular weight form of adenosine deaminase (ADA-S) is found. This suggestion is based on the assumption that the adenosine deaminase binding capacity is an inherent functional characteristic of adenosine deaminase-complexing protein. We report on the presence of adenosine deaminase-complexing protein immunoreactivity in mouse and rat determined with a species cross-reactive polyclonal anti-adenosine deaminase-complexing protein serum. In the mouse the tissue and subcellular distribution and the electrophoretic mobility in starch and polyacrylamide gels of the protein correspond with those of adenosine deaminase-complexing protein, but it does not bind the small molecular weight form of adenosine deaminase. Furthermore, in human, mouse, and rat kidney cortex adenosine deaminase and adenosine deaminase-complexing protein did not colocalize by immunohistochemistry. It is suggested that the function of adenosine deaminase-complexing protein is not adenosine deaminase-related.     

Simultaneous Single-Sample Determination of NMNAT Isozyme Activities in Mouse Tissues

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1) isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld^S) mouse. Wld^S is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld^S.     

Homocysteine in tissues of the mouse and rat

A method for the determination of L-homocysteine (Hcy) in tissues is described, which involves adsorption of adenosine and S-adenosyl-L-homocysteine (AdoHcy) in the tissue extract to dextran-coated charcoal, while leaving Hcy in solution. Sufficient dilution of the tissue homogenates and the presence of a reducing agent during the adsorption step are required to obtain high recovery of Hcy. Hcy is condensed with radioactive adenosine, and labeled AdoHcy is quantified by high performance liquid chromatography on a 3-micron reversed phase column. The amount of Hcy was determined in several tissues (liver, kidney, brain, heart, lung, and spleen) of mice and rats, and the concentrations of Hcy were in the range 0.5-6 nmol/g, wet weight. Hcy concentration was about 1 microM in mouse plasma. In mice, liver contained the highest amount of Hcy, and kidneys were also rich in Hcy. Similar concentrations were found in rat tissues. S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), the enzyme which is believed to catalyze the only pathway leading to Hcy formation in vertebrates, was nearly completely inactivated in mice injected with the drug combination 9-beta-D-arabinofuranosyladenine plus 2'-deoxycoformycin. This treatment induced a massive accumulation of AdoHcy in all tissues (Helland, S., and Ueland, P. M. (1983) Cancer Res. 43, 1847-1850). The amount of Hcy increased several-fold in kidney, whereas no change was observed in liver, heart, brain, lung, and spleen.     

Cyclic AMP binding protein from Jerusalem artichoke rhizome tissues

A cyclic AMP binding protein has been purified to electrophoretic homogeneity from Jerusalem artichoke rhizome tissues. Its MW is ca. 240 000 and the apparent constant of cyclic AMP binding to the protein is 2.3 × 10^?7 M. When tested using Millipore filter assay, cyclic AMP binding activity was enhanced by protamine and histone, but not by casein and phosvitin. Of several purine derivatives tested, only 5′-AMP and adenosine inhibited significantly the binding of cyclic AMP by the protein. The protein also binds adenosine and this binding is not affected by cyclic AMP or by other purine derivatives. The apparent binding constant for adenosine is 1.0 × 10^?6 M. The binding protein did not show protein kinase activity. In addition, it did not affect the chromatin-bound DNA dependent RNA polymerase of homologous origin, either in the presence or absence of cyclic AMP. The binding protein is devoid of the following activities: cyclic AMP phosphodiesterase, 5′-nucleotidase, adenosine deaminase and ATPase.     

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.     

A simple method for determining specific esterase activity in tissue extracts.

A simple procedure for estimating specificB-naphthyl esterase enzyme content and activity in small amounts of crude tissue extract is described. Esterase activity is determined by quantitative densitometry of histochemically stained acrylamide gels. Activity measured this way is linear with the extract volume applied. Esterase content is determined by isotopic labeling with a stoichiometric covalently binding enzyme inhibitor, diisopropylfluorophosphate; followed by electrophoresis and gel slice counting.     

Characterization of an insoluble adenosine deaminase complexing protein from human kidney

Evidence for the presence of an insoluble form of adenosine deaminase complexing protein in human kidney has been obtained. An initial study demonstrated that binding of monomeric adenosine deaminase to particulate material from kidney was saturable and could be blocked by preincubating the enzyme with soluble complexing protein. Treatment of particulate material with deoxycholate, followed by immunoassay of the detergent extract, confirmed the presence of an insoluble form of complexing protein in the kidney. Several other human organs examined by this technique contained smaller amounts of insoluble complexing protein. Complexing protein isolated from the soluble and particulate fractions of kidney homogenates were found to be structurally similar. The proteins had the same subunit M_r and showed complete crossreactivity with antiserum to soluble complexing protein. Indirect immunoperoxidase staining of renal cortical tissue revealed that complexing protein was concentrated in the brush border of the proximal tubules. These results indicate that (a) the soluble and insoluble forms of complexing protein from human kidney may be products of the same gene(s) and (b) a portion of the complexing protein in human kidney is bound to the brush border membranes of cells lining the proximal tubules.     

In Vitro and In Vivo Binding of S-Adenosyl-L-Homocysteine to Membranes from Rat Cerebral Cortex

Membranes from rat cerebral cortex are able to bind S-adenosyl-L-homocysteine (SAH) with a KD of 5 . 10(-7) M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2+ but not K+ and Na+. gamma-Aminobutyric acid, diazepine, noradrenaline and alpha antagonists are without any effect; S-adenosyl-L-methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

Studies of nicotinic acetylcholine receptor protein from rat brain: II. Partial purification

The pharmacological specificity of the binding of ¹²⁵I-labeled α-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the α-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12 600-fold has been obtained. Binding of ¹²⁵I-labeled α-bungarotoxin to this purified acetylcholine receptor protein is saturable with a K_d of 1·10^?8 M. Nicotine and acetylcholine iodide at concentrations of 10^?5 M inhibit ¹²⁵I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10^?4 M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10^?4 M. These data support the isolation of partially purified nicotinic acetylcholine receptor protein.     

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S-adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S-adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S-adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S-adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S-adenosylhomocysteine inhibit some transmethylation reactions.     

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...