Characterization of the phosphorylated form of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

Incubation of intact purified rat liver plasma membranes with insulin, cyclic AMP and ATP led to the activation of the peripheral "low-Km" cyclic AMP phosphodiesterase. When (gamma-32P]ATP was included in the incubation mixture, after purification of this enzyme to homogeneity it was found to contain 1 mol of alkali-labile 32P/mol of enzyme. Treatment of the homogeneous phosphorylated enzyme with alkaline phosphatase released all of the 32P from the protein while restoring its activity to the native state. The reversibility of the activation that is achieved by the phosphorylation of this enzyme could also be demonstrated with a high-speed supernatant from rat liver. This restored the activity of the activated membrane-bound enzyme to its native state. The Ka for the cyclic AMP-dependence of this process (1.6 micrometer) was unaffected by a range of ATP concentrations (1-10 mM) and by a range of membrane protein concentrations (0.2-2 mg/ml). Adenylyl imidodiphosphate could not substitute for ATP, and concanavalin A could not substitute for insulin, as essential ligands in the activation process. The purified activated enzyme exhibited Km 0.6 microM, Vmax 10.9 units/mg of protein and Hill coefficient (h) 0.47. The Vmax. for this activated enzyme was much higher than that of the native enzyme, yet h was much lower.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.     

The insulin- and glucagon-stimulated ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity.

The hormone-stimulated 'dense-vesicle' cyclic AMP phosphodiesterase was solubilized as a proteolytically 'clipped' species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native 'dense-vesicle' enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified 'clipped' enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].     

A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes.

1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.     

Effect of sodium deoxycholate on 5'-nucleotidase.

The 5'-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5'-nucleotidase exhibited the same properties as the 5'-nucleotidase in plasma membranes. The 5'-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and Km. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Isolation and characterization of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum

A new enzyme that hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate has been purified by a factor of 250 from the acellular slime mold Physarum polycephalum. Activity was assayed radioisotopically with [3H]Ap4A. Isolation of the enzyme was facilitated by dye-ligand chromatography. The enzyme symmetrically hydrolyzes Ap4A to ADP and exhibits biphasic kinetics for the substrate with values for the apparent Km of 2.6 micro M and 37 micro M. The two values of Vmax differ by a factor of 10. Mg2+, Ca2+, and other divalent cations inhibit the activity with 40-80% inhibition occurring at 0.5 mM. Mg2+, at 0.5 mM, decreases both values of Vmax by 50%, decreases the low Km value by about 30%, and increases the high Km value by about 100%. (Ethylenedinitrilo)tetraacetic acid (EDTA) and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), at 10 mM, inhibit the activity by 50%. ADP, ATP, Ap4, and Gp4 are equipotent inhibitors with 50% inhibition occurring at 30 micro M. AMP is a relatively weak inhibitor. The molecular weight of the enzyme is 26000 on the basis of elution of activity from a calibrated Sephadex G-75 column.     

Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography.

The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.     

Isolation and characterization of 5'-nucleotidase of a human pancreatic tumor cell line

5'-Nucleotidase of a human pancreatic tumor cell line (PaTu II) has been purified to homogeneity after extraction with detergent followed by two affinity chromatographic steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified 5'-nucleotidase revealed a single polypeptide band of 67 kDa. The Western blotted enzyme can be overlaid with concanavalin A proving its glycoprotein nature. After treatment with endoglycosidase F the deglycosylated 5'-nucleotidase exhibits an apparent molecular mass of 58 kDa. The kinetic properties of the solubilized enzyme have been determined (Km (AMP) of 4.0 microM; Vmax (AMP) = 8.6 muMOL/min.mg). Adenosine 5'-[alpha,beta-methylene]diphosphate is a competitive inhibitor of 5'-nucleotidase, whereas concanavalin A inhibits the enzymatic activity in a non-competitive manner. Polyclonal antibodies against purified 5'-nucleotidase of PaTu II have been produced which inhibit its enzymatic activity. Polyclonal antibodies raised against the enzyme purified from rat liver or bull seminal plasma also recognize 5'-nucleotidase of PaTu II cells, whereas polyclonal and monoclonal antibodies against the enzyme derived from chicken gizzard show no cross-reactivity. 5'-Nucleotidase appears to be concentrated in the plasma membrane of PaTu II cells as judged by cell fractionation and indirect immunofluorescence studies.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.     

Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver.

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.     

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflammatory drugs and progestins

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.     

Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.     

5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.     

2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein

Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes. Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons. The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme. 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl. Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM. Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of [3H]ACP to the purified enzyme preparation. The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP. These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.     

Purine biosynthesis de novo in bovine retina: purification and characterization of amidophosphoribosyl transferase and phosphoribosyl pyrophosphate synthetase.

The ability of bovine retina to synthesize purines de novo is shown for the first time. Amidophosphoribosyl transferase (EC 2.4.2.14), the enzyme controlling the rate of the process, and phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1), the enzyme regulating the intracellular contents of phosphoribosyl pyrophosphate (PRPP), were purified and characterized. The molecular masses of the enzyme subunits are similar to those of the purified enzyme from the liver. The molecular masses of amidophosphoribosyl transferase, PRPP synthetase catalytic subunit, and two PRPP synthetase-associated proteins are 50, 34, 39, and 41 kD, respectively. The apparent Km values of the enzymes and coenzymes are similar to those of the purified enzymes from the liver. For amidophosphoribosyl transferase, the apparent Km for Gln and PRPP are 0.75 +/- 0.05 and 0.66 +/- 0.09 mM, respectively (the corresponding Vmax values are 59 +/- 3 and 136 +/- 12 nmoles PPi/min per mg protein). For PRPP synthetase, the apparent Km for ribose-5-phosphate and ATP are 37.9 +/- 0.5 and 53 +/- 7 microM, respectively (the corresponding Vmax values are 61 +/- 4 and 52 +/- 3 nmoles PRPP/min per mg protein). The sensitivity of the retinal PRPP synthetase to inhibition by ADP and AMP was significantly lower than that of the enzyme from the liver.     

Isolation of beta-N-acetylhexosaminidase from rabbit semen and its role in fertilization.

Beta-N-Acetylhexosaminidase was purified from the rabbit seminal plasma by a three-step procedure involving hydroxyapatite, Sephadex G-200 and concanavalin A--Sepharose chromatography. The specific activity of the purified preparation was 56mu mol/min per mg of protein, which represented a 226-fold purification and a 54% yield of the enzyme activity. The purified enzyme was electrophoretically homogeneous. The homogeneous enzyme showed optimal activity at pH4.0. The apparent Km value and Vmax. were 1.4 mM and 56mu mol/min per mg of protein respectively. Metal ions such as Ag + and Hg2+ and p-chloromercuribenzoate strongly inhibited the enzyme activity. The treatment of rabbit ova with a mixture of Beta-N-acetylhexosaminidase and arylsulphatase A results in the swelling of the zona pellucida.     

Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.     

beta-Alanine synthase: purification and allosteric properties.

beta-Alanine synthase has been purified greater than 1000-fold to homogeneity from rat liver. The enzyme has a subunit molecular weight of 42,000 and a native size of hexamer. The enzyme undergoes ligand-induced changes in polymerization: association in response to the substrate, N-carbamoyl-beta-alanine, and the inhibitor, propionate; and dissociation in response to the product, beta-alanine. The ability of the substrate to associate the pure native enzyme to a larger polymeric species was exploited in the final purification step. The purified enzyme had a pI of 6.7, a Km of 8 microM, and a kcat/Km of 7.9 x 10(4) M-1 s-1. Positive cooperativity was observed toward the substrate N-carbamoyl-beta-alanine, with nH = 1.9. Such cooperativity occurred at substrate concentrations below 12 nM, so that this activation most likely occurs at a regulatory site, with a significantly stronger affinity for N-carbamoyl-beta-alanine than that shown by the catalytic site. The enzyme was sensitive to denaturation, which could be minimized by avoiding heat steps during the purification and by the presence of reducing agents. Such denatured enzyme had little change in Vmax, but had much higher Km, and had also lost the ability to associate or dissociate in response to effectors. After purification, enzyme stability was achieved by the addition of glycerol and detergent.