Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.     

Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I

The mechanism of Ca2+ inhibition of carbamylphosphate synthetase I has been investigated using purified enzyme obtained from livers of rats fed a high protein diet. Binding of Mn2+ to the enzyme was measured by EPR techniques at pH 7.8, and Scatchard plots of the data indicated one Mn2+-binding site with a K'd of 13 microM. From competition studies between Mn2+ and Ca2+ or Mg2+ binding, values of 180 microM were obtained for K'd (Mg) and 193 microM for K'd (Ca). A nonlinear least squares curve fitting program was used to calculate the K'm for MgATP2- at the metal-nucleotide binding sites using a simplified rate equation of the enzyme reaction mechanism. Values of 140 and 2420 microM were obtained for K'm (MgATP) at the first and second sites, respectively, at pH 7.8, with a free Mg2+ of 1 mM and other substrates and activators present at saturating concentrations. Variations of the bicarbonate, N-acetylglutamate, and ammonia concentrations in the absence and presence of different amounts of total calcium, from which free Ca2+, free Mg2+, MgATP2-, and CaATP2- concentrations were calculated, permitted values for K'i (CaATP) to be obtained by graphic procedures. Mean values of 375 and 120 microM were obtained for K'i (CaATP) at the first and second sites, respectively. Using the above kinetic constants, a computer model of the enzyme reaction was constructed and tested using two further sets of kinetic data obtained by varying the concentrations of Mg2+, Ca2+, MgATP2-, and CaATP2-. Poor fits were obtained unless the formation of a mixed complex involving CaATP2- competition with MgATP2- at the second metal-nucleotide-binding site was incorporated into the rate equation. Nonlinear least squares curve fitting of both sets of experimental data gave a well determined value of 124 microM for this final CaATP2- inhibitory constant. Sensitivity tests for variation of the primary kinetic constants with the computer model showed that the inhibitory effect of free Ca2+ was weak and that the observed calcium inhibition of carbamylphosphate synthetase can be accounted for primarily by competitive interaction of CaATP2- at the second MgATP2- binding site. With 1 mM free Mg2+ and 5 mM MgATP2-, half-maximal inhibition of enzyme activity was obtained with 0.2 mM CaATP2-.     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

Turkey acrosin. I. Isolation, purification, and partial characterization

Acrosin was extracted from turkey spermatozoa by use of urea together with sonication and freezing, and purified approximately 18-fold by sequential use of chromatofocusing and affinity chromatography. The use of chromatofocusing for the initial purification step proved to be superior to preparative isoelectric focusing. Similar to acrosin from many mammalian species, turkey acrosin was found to be a glycoprotein possessing characteristics of serine proteases. Polyacrylamide gel electrophoresis (PAGE) of the enzyme indicated the presence of two isozymes. Sodium-dodecyl sulfate PAGE under reducing conditions revealed three subunits with approximate molecular weights of 11,700, 13,900, and 15,900.     

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.     

Expression,purification, and characterization of human tyrosyl-tRNA synthetase

Human tyrosyl-tRNA synthetase is a homodimeric enzyme and each subunit is near 58 KD. It catalyzes the aminoacylation of tRNA(Tyr) by L-tyrosine. The His(6)-tagged human TyrS gene was obtained by RT-PCR from total RNA of human lung giant-cell cancer strain 95 D. It was confirmed by sequencing and cloned into the expression vector pET-24 a (+) to yield pET-24 a (+)-HTyrRS, which was transfected into Escherichia coli BL21-CodonPlus-RIL. The induced-expression level of His(6)-tagged human TyrRS was about 24% of total cell proteins under IPTG inducing. The recombinant protein was conveniently purified in a single step by metal (Ni(2+)) chelate affinity chromatography. About 22.3mg purified enzyme could be obtained from 1L cell culture. The k(cat) value of His(6)-tagged human TyrRS in the second step of tRNA(Tyr) aminoacylation was 1.49 s(-1). The K(m) values of tyrosine and tRNA(Tyr) were 0.3 and 0.9 microM. Six His residues at the C terminus of human TyrRS have little effect on the activities of the enzyme compared with other eukaryotic TyrRSs.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

Acid nucleoside triphosphatase: partial purification and characterization of a new enzyme from human serum

Acid nucleoside triphosphatase (Acid NTPase), an enzyme which catalyzes the hydrolysis of all nucleoside triphosphates to the corresponding diphosphates was purified from human serum with a purification factor of 190 and a recovery of 31%. The molecular weight was 75,000 as estimated by gel filtration. Gel-electrophoresis revealed an Rf-value of 0.11, and the isoelectric point was determined at pH 4.4. It exhibited a temperature optimum of 44 degrees C and the activation energy was estimated to be 41.6 kJ/mol. The enzyme was active in the absence of divalent cations, since activity was not inhibited by EDTA. The presence of this chelator reduced the Km-value from 70 to 40 microM. Inhibitor experiments revealed that tartrate was a weak mixed-type noncompetitive inhibitor, Ki = 88 mM. The enzyme was specific for the hydrolysis of nucleoside triphosphates. P-nitrophenyl phosphate was not accepted as a substrate. The enzyme revealed optimum activity at the exceptionally acid pH of 3.0. These unique characteristics indicate the presence of a novel enzyme.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

Phosphodiesterase I in human urine: purification and characterization of the enzyme

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.     

Purification and partial characterization of alpha-N-acetylglucosaminidase from human liver.

A deficiency in alpha-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39,000-fold from liver to homogeneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepharose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl alpha-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50 degrees C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of alpha-N-acetylglucosaminidase.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Biosynthesis of monoterpenes: partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis.

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage (Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl₂ (1 mm). ZnCl₂ was nearly as effective as MnCl₂, and MgCl₂ could substitute for MnCl₂ only at tenfold higher concentrations. The apparent K_m and V of the enzyme were 10^?5m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10^?4m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10^?5m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.