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.     

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.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Identity of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase

Identification of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase has been done. When a mitochondria extract was subjected to isoelectric focusing, the two enzyme activities were identically focused. This procedure and DEAE-Sepharose chromatography revealed multiple forms of the enzyme, in which the main form was purified. In the various purification steps the two enzyme activities appeared in the same fraction. The enzyme of the final preparation step gave a single band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. During the purification, a similar increase of the specific activity and yield were obtained in the two activities. Phenylalanine was found to be a competitive inhibitor of asparagine transaminase. These results suggest the identity of the two enzymes.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Choline acetyltransferase

No multiple forms of choline acetyltransferase were found in extracts of human, mouse, rabbit, guinea pig, cat, and rat brain. A single form of this enzyme only was also demonstrated in bovine nervous tissue, including brain, dorsal and ventral roots, spinal cord, and femoral nerve. The difference from other published findings is believed to be due to ammonium sulfate fractionation, which was not used in the present study. In addition, multiple forms of the enzyme were obtained by others using isoelectric focusing, whereas this study employed gel filtration. Choline acetyltransferase was highly purified from mouse brain using a procedure similar to that used for the enzyme from bovine brain. The steps involved: (1) making an acetone-chloroform powder from whole mouse brains, (2) extracting the powder and chromatographing the soluble fraction with organomercurial Sepharose, (3) passing the enzyme solution through a column of DEAE-cellulose, (4) eluting from hydroxyapatite, and (5) removing contaminants by subunit exchange chromatography. The final preparation was essentially homogeneous as revealed by polyacrylamide gel electrophoresis.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.     

Resolution and purification of taurine- and GABA-synthesizing decarboxylases from calf brain

The present work describes a procedure for the co-purification of cysteine sulfinate decarboxylase (CSAD) and glutamate decarboxylase (GAD) from calf brain. A crude enzyme preparation was first made from brain homogenate by acid precipitation and ammonium sulphate fractionation. Subsequent fractionation of the decarboxylase preparation by cation exchange chromatography on CM-Sepharose CL-6B revealed the existence of a specific CSAD enzyme, which has no GAD activity. The GAD activity peak was found to possess CSAD activity. Further fractionation by gel filtration on Sephacryl S-200 separated the specific CSAD activity into two enzyme forms, one of them having a molecular weight of 150,000 and the other of 71,000. GAD activity was eluted from the gel filtration column in a single peak (mol wt 330,000) and showed CSAD activity. The purification of the specific CSAD enzyme was 920-fold and that of GAD activity 850-fold as compared with the starting material, whole calf brain. SDS gel electrophoresis indicated that the purified CSAD and GAD enzymes consisted of two or more subunits. The crude decarboxylase preparation was analysed by isoelectric focusing in ultra-thin polyacrylamide gel in the pH range 3.5-10.0. The most active fraction of CSAD indicated an isoelectric point of 6.5 and that of GAD 6.8. The pH optimum for CSAD activity in the crude preparation was 7.2 and that for GAD activity 7.9.     

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.     

Purified guanylate cyclase: characterization, iodination and preparation of monoclonal antibodies

Guanylate cyclase was purified from the soluble fraction of rat lung using a modification of procedures published previously. The purified enzyme exhibited specific activities, at pH 7.6, of 219-438 nmoles/mg protein/min and 34-60 nmoles/mg protein/min with Mn2+ and Mg2+ as cation cofactors, respectively. The specific activity changed as a function of the protein concentration due to a change in Vmax with no alteration of the Km for GTP. The enzyme migrated as a single band coincident wih guanylate cyclase activity on nondenaturing polyacrylamide and isoelectric focusing gels (isoelectric point = 5.9). Purified guanylate cyclase had an apparent molecular weight of 150,000 daltons as determined by gel filtration chromatography and polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate revealed a single subunit of 72,000 daltons, suggesting that the enzyme is a dimer of an identical subunit. The purified enzyme could be activated by nitric oxide, indicating that this compound interacts directly with the enzyme.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

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.     

Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli

An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatography. The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 mumol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 g enzyme. Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61,000 and 35,000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-Mr fragment. Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Purification and properties of biliverdin reductases from pig spleen and rat liver.

Biliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34,000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxiodoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 microM while that for NADH was 1.5-2 mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 microM whereas that in the NADH system was 1-2 microM. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IX alpha-biliverdin was the most effective substrate among the four biliverdin isomers, and the dimethylester of IX alpha-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34,000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IX alpha-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.