Dissociation and reconstitution of human ferroxidase II.

The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.     

Evolution of allantoinase and allantoicase involved in urate degradation in liver peroxisomes. A rapid purification of amphibian allantoinase and allantoicase complex, its subunit locations of the two enzymes, and its comparison with fish allantoinase and allantoicase

Allantoinase and allantoicase are located in the same protein molecule in amphibian liver, whereas the two enzymes are different proteins in marine fish and invertebrate liver (Takada, Y., and Noguchi, T. (1983) J. Biol. Chem. 258, 4762-4764). The amphibian enzyme was rapidly purified from frog liver by using its following characteristics. 1) The enzyme binds to the intracellular membranes in the hypotonic solution. 2) The membrane-bound enzyme is not solubilized by the detergent. 3) The membrane-bound enzyme is solubilized by oxaloacetate. The electrophoresis of the purified enzyme gave a single protein band in the absence of sodium dodecyl sulfate, and gave two protein bands with molecular weights of 48,000 and 54,000, respectively, in the presence of sodium dodecyl sulfate. With a specific antibody raised against each subunit, allantoinase activity was found to be from the large subunit, and allantoicase activity to be from the small subunit. This amphibian allantoinase and allantoicase complex was compared with allantoinase and allantoicase purified from fish liver. Fish allantoinase was a single peptide and fish allantoicase was composed of two identical subunits. Fish allantoinase had an identical molecular weight with amphibian large (allantoinase) subunit and the subunit of fish allantoicase with amphibian small (allantoicase) subunit. These results suggest that the evolution of fish to amphibian resulted in the dissociation of allantoicase into subunits and in the association of allantoinase with allantoicase. The two enzymes are lost by further evolution.     

Turnover of ammonium-inducible glutamate dehydrogenase during induction and its rapid inactivation after removal of inducer from Chlorella sorokiniana cells.

When ammonia was removed from Chlorella sorokiniana cells, which contain an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH), the activity of this enzyme decayed with a half-life of approximately 8 min. By use of rocket immunoelectrophoresis, indirect immunoprecipitation, and indirect immunoadsorption (coupled with pulse-chase experiments with 35S-labeled sulfate), the rapid initial loss in activity was shown to be due to enzyme inactivation rather than degradation of NADP-GDH antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained with anti-NADP-GDH immunoglobulin G showed that enzyme inactivation is accompanied by the conversion of enzyme subunits (Mr = 59,000) to a protein with a molecular weight of 118,000. Because this protein was stable during boiling and in the presence of sodium dodecyl sulfate and high concentrations of mercaptoethanol or dithiothreitol, it was tentatively assumed to be a covalently linked dimer of enzyme subunits. Pulse-chase experiments showed that total NADP-GDH antigen was subject to rapid degradation (t 1/2 = 88 min) in induced cells, and the same degradation rate was maintained after removal of ammonia from induced cells.     

Induction of rat hepatic alkaline phosphatase and its appearance in serum: electrophoretic characterization of liver-membranous and serum-soluble forms

Simultaneous bile duct ligation and colchicine injection (2 mg/kg body weight) in rats caused a remarkable induction of alkaline phosphatase in the liver. Concomitantly, a marked elevation of the enzyme activity occurred in the serum, and three activity peaks (peaks I, II, and III) were separated by Sephadex G-200 gel filtration. By several criteria for alkaline phosphatase isoenzymes it was determined that the liver-derived enzyme was distributed in peak I (30% of total serum activity) as a vesicle-bound form and in peak II (65%) as a soluble form, while the intestinal enzyme was contained in peak III (5%). The serum alkaline phosphatase in peaks I and II was compared with the liver enzyme extracted from plasma membrane with n-butanol. Under non-reducing conditions, the soluble form of peak II showed an electrophoretic mobility different from that of the liver enzyme; in the presence of sodium dodecyl sulfate the serum-soluble form migrated a little more slowly than the liver one, while in the presence of Triton X-100 the former migrated much faster than the latter. The sedimentable fraction of peak I was found to contain two forms corresponding to the serum-soluble and liver-membranous forms. Neuraminidase treatment of these two forms reduced their mobilities but did not abolish the relative difference in their mobilities on gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulfate. Under reducing conditions, however, each form (which was dissociated into single subunits) migrated with an identical mobility on sodium dodecyl sulfate gel electrophoresis. These results suggest that the hepatic alkaline phosphatase exists as conformationally different forms in the serum and the liver membrane (even solubilized), but the difference is no longer preserved after their denaturation into subunits.     

Nicotinamide adenine dinucleotide dependent isocitrate dehydrogenase from beef heart: subunit heterogeneity and enzyme dissociation.

Heterogeneity in the subunits of nicotinamide adenine dinucleotide dependent isocitrate dehydrogenase from beef heart mitochondria was investigated using one- and two-dimensional electrophoretic analyses in polyacrylamide gels. Electrophoresis under nondenaturing conditions, at several values of pH and gel concentration, followed by second-dimension electrophoresis in the presence of sodium dodecyl sulfate showed that the active enzyme contains four different subunits. The details of these two-dimensional patterns, reelectrophoresis of the active enzyme band under nondenaturing conditions, together with additional evidence indicate that under certain nondenaturing conditions the enzyme exists partially dissociated into its subunits. The molecular weights of the four subunits, determined from electrophoretic mobilities obtained in the presence of sodium dodecyl sulfate, were different, varying between 39 000 and 41 300. Tryptic peptide maps of the subunits are substantially different.     

Isolation and characterization of a manganese-containing superoxide dismutase from yeast.

The cyanide-insensitive superoxide dismutase of yeast has been shown to be localized in the mitochondrial matrix. This enzyme has been isolated in good yield from bakers' yeast. Its molecular weight is 96,000. It is a tetramer, being composed of four subunits of equal size. Exposure to sodium dodecyl sulfate at 100 degrees caused dissociation into dimers, while similar treatment but in the presence of 2-mercaptoethanol caused complete dissociation into monomers. This enzyme contains 1 atom of manganese per subunit and its absorption in the visible suggests Mn(III) in the resting enzyme. Ascorbate caused partial bleaching, presumably by reduction to Mn(II). The amino acid composition was determined. This enzyme has activity comparable to that of other previously reported superoxide dismutases and like the chicken mitochondrial and the bacterial enzymes, its rate of reaction with O2 falls as the pH is raised above 7.8. Crystals of high quality were easily prepared.     

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.     

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.     

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.     

Molecular structure of aminopeptidase A from bovine kidney

Using gel-filtration through Sephadex G-100 and polyacrylamide gel electrophoresis in the presence of 0,5% sodium dodecyl sulfate, it was found that aminopeptidase A has a molecular weight of 65 000 +/- 2000 and is made up of two subunits with mol. weights of 33 000 +/- 2000. Each subunit consists of two polypeptide chains with mol. weights of 22 000 +/- 2000 and 12 000. During enzyme dissociation into subunits the aspartylnaphtylamidase activity is lost, while the glutamylnaphtylamidase activity is retained.     

Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain.

This report describes the purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity. Criteria for purity include sodium dodecyl sulfate and disc gel electrophoresis, as well as isoelectric focusing (pI = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide, which, upon hydrolysis of the naphthylamide, results in the appearance of the fluorescent product, beta-naphthylamine (beta NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 mumol of beta NA min-1 mg-1. The only peptide product formed was shown to be L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH was shown to yield L-pyro-glutamyl-L-histidyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis indicated that the enzyme consists of a single polypeptide chain having molecular weights of 70,000 and 73,500, respectively. Rat brain TRH deamidase has an apparent Km of 34 micron, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-beta-naphthylamide as a substrate. With this substrate, TRH was shown to be a competitive inhibitor with an apparent Ki of 120 +/- 20 micron.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

gamma-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity.

gamma-Glutamylcysteine synthetase was purified from rat liver by an improved method involving chromatography on Sepharose-aminohexyl-ATP to a specific activity of about 1600 units/mg, or approximately twice that previously obtained; it is thus the most active preparation of this enzyme thus far isolated. The earlier preparation, which is homogeneous on polyacrylamide gel electrophoresis, exhibits "half of the sites" reactivity in that it binds a maximum of 0.5 mol of the inhibitor L-methionine-S-sulfoximine phosphate per mol of enzyme. In contrast, the present enzyme preparation binds 1 mol of methionine sulfoximine phosphate per mol of enzyme; it also differs from the enzyme obtained earlier in exhibiting much less ATPase activity and less activity in catalyzing ATP-dependent cyclization of glutamate. gamma-Glutamylcysteine synthetase dissociates in sodium dodecyl sulfate into two nonidentical subunits of apparent molecular weights 74,000 and 24,000; after cross-linking with dimethyl-suberimidate, a species having a molecular weight of about 100,000 was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. New information has been obtained about the interaction of the enzyme with glutamate analogs; thus, the enzyme is active with such glutamate analogs as beta-glutamate, N-methyl-L-glutamate, and threo-beta-hydroxy-L-glutanate, and it is effectively inhibited by cis-1-amino-1,3-dicarboxycyclonexane, 2-amino-4-phosphonobutyrate, and gamma-methylglutamate.     

Latent proteinase activity of gamma-glutamyl transpeptidase light subunit.

gamma-Glutamyl transpeptidase, which is composed of two unequal subunits, exhibits proteinase activity when treated with agents such as urea and sodium dodecyl sulfate. The heavy subunit is preferentially and rapidly degraded. The enzyme also degraded bovine serum albumin in the presence of urea; however, several other proteins and model proteinase substrates were not cleaved. Treatment of the enzyme with 6-diazo-5-oxo-L-norleucine, a gamma-glutamyl analog, results in parallel loss of transpeptidase and proteinase activities indicating that the site at which gamma-glutamylation of the enzyme occurs (presumably a hydroxyl group on the light subunit) is also involved in proteinase activity. The purified light subunit, but not the heavy subunit, exhibits proteinase activity even in the absence of urea. Results suggest that dissociation of the enzyme unmasks the proteinase activity of the light subunit involving the site at which gamma-glutamylation of the enzyme occurs, and that the heavy subunit may impose transpeptidase reaction specificity by contributing the binding domains for gamma-glutamyl substrates.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association.

The HLA-linked B cell alloantigen (p29,34) is composed of two subunits of 29,000 (p29) and 34,000 (p34) molecular weight. The partially purified HLA-linked B cell alloantigen was purified by a final step of preparative sodium dodecyl sulfate-gel electrophoresis. An antiserum was prepared against p29,34 which specifically lysed B lymphocytes. In sodium dodecyl sulfate at 21 degrees, p29 and p34 remained noncovalently associated and retained immunologic activity; subunit dissociation at higher temperatures correlated with loss of immunologic activity. Although the pI values of p29 and p34 are 6.1 and 5.2, respectively, the subunits co-electrofocus under nondenaturing conditions. In addition, cross-linking studies showed the B cell antigen has a (p29)1(p34)1 subunit structure.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Purification and some properties of a neutral muscle pyrophosphatase.

In the water-soluble fraction of rabbit skeletal muscle, at least two types of inorganic pyro phosphatase (PPase) are distinguishable on ion exchange column chromatography. One of them, pyrophosphatase-A (PPase-A), was isolated in an electrophoretically homogeneous form. This enzyme catalyzed the hydrolysis of PPi but not that of other phosphate esters. Only Mg2+ was required for activity and stability. Other cations such as Ca2+, Co2+, Mn2+, and Zn2+ had no activating effect. The activity of this PPase was optimum at pH 7.4. ATP, ADP, sodium imidodiphosphate (PNP), p-chloromercuribenzoate, and Ca2+ inhibited its enzymic activity. The enzyme was protected by dithiothreitol (DTT) against heat denaturation. The molecular weight was estimated to be 67,000 by gel filtration and the molecular size of the subunit was found to be 35,000 by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme probably consists of two identical subunits of 35,000 daltons.     

Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.