The biochemical basis for the conjugation of bile acids with either glycine or taurine

All animals, except for the placental mammals, conjugate their bile acids exclusively with taurine. However, in certain of the placental mammals, glycine conjugates are also found. The basis for the appearance of glycine conjugation among the placental mammals was investigated. The reaction of choloyl-CoA with glycine and taurine, as catalysed by the soluble fraction from guinea-pig liver, had a high affinity for taurine and a poor affinity for glycine. The predominant synthesis of glycine conjugates in the guinea pig can be related to the fact that guinea-pig liver contains an unusually low concentration of taurine and a high concentration of glycine. Rabbits make exclusively glycine conjugates and their livers also contain low concentrations of taurine. However, the biochemical basis for their glycine conjugation is more straightforward than in the guinea pig in that the soluble fraction from rabbit liver has a high affinity for glycine and a poor affinity for taurine. Alternative-substrate-inhibition studies with glycine and taurine in soluble fractions from guinea-pig and rabbit liver revealed that glycine and taurine were mutually inhibitory. This suggests that there is only one enzyme for glycine and taurine conjugation in these tissues. The soluble fractions from bovine liver and human liver also made both glycine and taurine conjugates and evidence is presented that suggests that there is only one enzyme in these tissues too. Even the rat, which excretes mostly taurine conjugates, could make both glycine and taurine conjugates in vitro. However, in contrast with all of the placental mammals studied, the supernatant fraction from liver of the chicken, and other non-mammals, could not make glycine conjugates even in the presence of very high concentrations of glycine.     

The co-purification and common identity of cholyl CoA:glycine- and cholyl CoA:taurine-N-acyltransferase activities from bovine liver.

A procedure for the purification of cholyl CoA:glycine and taurine N-acyltransferase activities from the soluble cell fraction of bovine liver is described. The procedure results is an 900-fold enrichment relative to the soluble cell fraction. The final preparation gives a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mr = 50,900, and runs as a single peak, Mr = 47,000, on gel filtration. The preparation is approximately 80% pure as judged by isoelectric focusing and focuses at a pH of 6.6. The glycine and taurine conjugating activities co-purified and did not separate to any extent in any of the chromatographic steps employed, including a gradient elution from an affinity column and an isoelectric focusing column. Also, kinetic analysis revealed that glycine and taurine appear to compete for a common active site. The two activities had identical temperature-denaturation curves and were equivalently stabilized against temperature denaturation by taurocholate. This data provides strong evidence for a common enzyme for both glycine and taurine conjugation in bovine liver. A preliminary kinetic characterization of the enzyme revealed non-Michaelis-Menten kinetics.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Enzymes that metabolize acyl-coenzyme A in the monkey--their distribution, properties and roles in an alternative pathway for the excretion of nitrogen.

1. In various tissues from the monkey (Macaca fuscata), acyl-coenzyme A (CoA) hydrolase activities were found to be widely distributed within a 2-10 times range and present in liver cytosol having mol. wt of ca 60,000. 2. Acyl-CoA: amino acid N-acyltransferase activity were 4-250 times higher in liver and kidney than in other tissues, even no activity in heart, lung, and plasma. 3. The transferases abounded in liver mitochondria, being distributed evenly between the intracristate space, the inner membrane, and the matrix. 4. The partially purified transferases with benzoyl-CoA or phenylacetyl-CoA as substrates were shown to have mol. wt of ca 30,000 and reacted only with glycine or L-glutamine, respectively. 5. No amino acid tested had any effects on the enzyme as either inhibitors or activators. 6. These results suggest that the enzymes that metabolize acyl-CoA constitute an alternative pathway for the excretion of nitrogen.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

A kinetic comparison of partially purified rat liver Golgi and rat serum galactosyltransferases.

UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase was partially purified from rat liver Golgi membranes and rat serum. The kinetic parameters of the two enzymes isolated by affinity chromatography were compared with each other and with those for commercial bovine milk galactosyltransferase. When N-acetyl-glucosamine was the acceptor the Km values for UDP-galactose were 65,52 and 43 microM for the rat liver Golgi, rat serum and bovine milk enzymes respectively. The Km values for N-acetylglucosamine were 0.33, 1.49 and 0.5 mM for the three enzymes respectively. The Km values for UDP-galactose, with glucose as acceptor in the presence of 1 mg of alpha-lactalbumin, were 23, 9.0 and 60 microM for the three enzymes respectively, and the Km values for glucose were 2.3, 1.8 and 2.0 mM respectively. The effects of alpha-lactalbumin in both the lactosamine synthetase and lactose synthetase reactions were similar. The activation energies were 94.0 kJ/mol (22.5 kcal/mol) and 96.0 kJ/mol (22.9 kcal/mol) for the Golgi and serum enzymes respectively. Although some differences in Km values were observed between the rat liver Golgi and serum enzymes, the values obtained suggest a high degree of similarity between the kinetic properties of the three galactosyltransferases.     

Properties and subcellular distribution of two partially purified ornithine transcarbamoylases in cell suspensions of sugarcane

The spatially separated forms of ornithine transcarbamoylase (EC 2.1.3.3) of different molecular weights coexist in sugarcane (Saccharum sp.). The smaller form of the enzyme (mol wt 79,000) appears to be cytoplasmic, while a larger form (mol wt 224,000) sedimented with mitochondria. The Km of the cytoplasmic enzyme for ornithine was 3.11 mm, while the enzyme in the mitochondrial fraction had a Km of 0.50 mm for this substrate; both enzymes had similar affinity for carbamoyl phosphate (0.12 mm). Characteristics of the smaller ornithine transcarbamoylase are in keeping with a predominantly catabolic function, those of the enzyme which sediments with mitochondria, with an anabolic function. Only the mitochondrial enzyme was regulated in vivo by exogenous arginine.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Purification and properties of aminoacetone synthetase from beef liver mitochondria

Aminoacetone synthetase from beef liver mitochondria was purified to homogeneity and shown to be a member of the pyridoxal 5'-phosphate-dependent family of enzymes. This enzyme catalyzes the condensation of glycine and acetyl-CoA to produce CO2, CoA, and the stable product aminoacetone. Bovine aminoacetone synthetase is a dimer (Mr 56,000) of identical subunits and contains 2 mol of pyridoxal phosphate/mol of dimer. The holoenzyme was resolved by dialysis against cysteine and has a pI of 5.2. The holoenzyme shows an absorption maximum at 428 nm which undergoes a shift to 335 nm when reduced with sodium borohydride. The Km values of glycine and acetyl-CoA were 22 mM and 53 microM, respectively. Initial velocity studies indicate that the condensation reaction proceeds by an ordered mechanism. With the exception of aminomalonate, bovine aminoacetone synthetase acts specifically on glycine and acetyl-CoA. Coupled reactions of purified bovine aminoacetone synthetase and porcine L-threonine dehydrogenase demonstrated the interconversion of threonine and glycine.     

Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas haloplanktis

Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-(32)P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4-8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K(m) 0.26 mM) and γ-glutamylcysteine (K(m) 0.25 mM); a lower affinity was measured for glycine (K(m) 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K(i) 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol(-1)) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol(-1). To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.     

Purification and physicochemical properties of NADPH-specific dihydropteridine reductase from bovine and human livers

A new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahydropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65,000-70,000. The enzyme was composed of two subunits with identical molecular weight of 35,000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.     

Isolation and characterization of dihydropteridine reductase from human liver.

Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.     

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

Rat liver bile acid CoA:amino acid N-acyltransferase: expression,characterization, and peroxisomal localization

Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver.     

ACETYLCHOLINESTERASE IN MOUSE BRAIN, ERYTHROCYTES AND MUSCLE

Abstract— The properties of the enzyme acetylcholinesterase (EC 3.1.1.7) from mouse brain, erythrocytes and muscle were investigated. The enzymes were examined by gel filtration in Sephadex G-200, polyac-rylamide gel electrophoresis, immunological reactions, active-site labelling with tritiated di-isopropyl-phosphorofluoridate and also their kinetic properties were compared. All three enzymes appeared to have a single active small mol. wt. component of 80,000 to 82,000 which produced higher mol. wt. forms by aggregation. The partial purification of the enzyme from brain was achieved by affinity chromatography and this product was used to prepare antibodies. The purified immunoglobulin was shown to react with all three enzymes.     

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus

1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.     

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.     

2,4-Dienoyl coenzyme A reductases from bovine liver and Escherichia coli. Comparison of properties

2,4-Dienoyl-CoA reductases, enzymes of the beta-oxidation of unsaturated fatty acids which were purified from bovine liver and oleate-induced cells of Escherichia coli, revealed very similar substrate specificities but distinctly different molecular properties. The subunit molecular weights, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 32,000 and 73,000 for the mammalian and the bacterial enzyme, respectively. The native molecular weights, calculated from sedimentation coefficients and Stokes radii yielded 124,000 for the bovine liver and 70,000 for the bacterial enzyme. Thus, bovine liver 2,4-dienoyl-CoA reductase is a tetramer consisting of four identical subunits. The E. coli 2,4-dienoyl-CoA reductase, however, possesses a monomeric structure. The latter enzyme contains 1 mol of FAD/mol of enzyme, whereas the former reductase is not a flavoprotein. The bovine liver reductase reduced 2-trans, 4-cis- and 2-trans,4-trans-decadienoyl-CoA to 3-trans-decenoyl-CoA. The E. coli reductase catalyzed the reduction of the same two substrates but in contrast yielded 2-trans-decenoyl-CoA as reaction product. Certain other properties of the two 2,4-dienoyl-CoA reductases are also presented. The localization of the reductase step within the degradation pathway of 4-cis-decenoyl-CoA, a metabolite of linoleic acid, is discussed.     

Adenosine 3'':5''-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.