L-fucose metabolism in mammals. Purification of pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase by NAD+-Agarose affinity chromatography.

Pork liver has previously been reported to contain a soluble enzymatic pathway which converts L-fucose to 2-keto-3-deoxy-L-fuconate and D-arabinose to 2-keto-3-deoxy-D-arabonate. We now report the isolation from pork liver of a soluble NAD+-dependent dehydrogenase which acts on both 2-keto-3-deoxy-L-fuconate and 2-keto-3-deoxy-D-arabonate. This enzyme has been purified to homogeneity by a five-step procedure; the final step involved affinity chromatography on NAD+-agarose. A purification factor of about 3000-fold was achieved with a yield of over 20%. The enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 9.1 and 7.0 and on the basis of sedimentation equilibrium analysis with the ultracentrifuge. The molecular weight of the native enzyme is about 100,000 while disc gel electrophoresis in the presence of sodium dodecyl sulfate and thiol showed the presence of a polypeptide of molecular weight 26,800; these results suggest that the enzyme is a tetramer. The enzyme has an isoelectric point of 5.4. The enzyme is unstable in the dilute state and in the absence of thiol but can be kept for 2 years at -70 degrees at a protein concentration of 4 mg per ml and in the presence of 1 mM dithiothreitol.     

The molecular weight of pig liver microsomal carboxylesterase

The enzyme carboxylesterase, isolated from the microsomes of pig liver, was found to have a molecular weight of 180,000 in dilute salt solutions as determined by the method of sedimentation equilibrium. In the presence of 6 m guanidine hydrochloride, 0.1 M β-mercaptoethanol, the molecular weight, uncorrected for preferential solvation, was found to be 61,000, also by the method of sedimentation equilibrium. The molecular weight determined for the enzyme (reduced and alkylated with acrylonitrile) in 6 m guanidine hydrochloride by the method of analytical gel chromatography was found to be 58,200. The method of disc gel electrophoresis in sodium dodecyl sulfate yielded a molecular weight of 62,000. The conclusion of the study is that the native carboxylesterase molecule is comprised of three subunits each with a molecular weight of approximately 60,000.     

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.     

Differences in plasma membrane organization of neuroblastoma cells grown in the differentiated and undifferentiated states.

Rabbit skeletal muscle creatine kinase is inactivated when stored at ?17 °C in the presence of either chloride or nitrate. Other anions are not effective. Associated with the inactivation is an altered electrophoretic mobility and the loss of four out of the eight titratable thiol groups in the dimeric catalytic protein of molecular weight 82,600. The altered inactive form is separated from the native active enzyme by electrofocusing, and its catalytic activity is restored by treatment with 2-mercaptoethanol. Gel electrophoresis in the presence and absence of 2-mercaptoethanol establishes that solutions of the inactive enzyme are heterogeneous, containing mostly a protomeric polypeptide of molecular weight 41,000, but also significant amounts of disulfide-linked dimers, trimers, and tetramers. Sedimentation equilibrium analysis confirms the existence of higher molecular weight aggregates along with the preponderant protein species of molecular weight 43,000.     

Purification and properties of Oxidized and reduced forms of Hepatic fructose 1,6-bisphosphatase

D-Fructose 1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) was isolated from rat liver in two forms: "A," isolated in the presence, and "B," isolated in the absence of dithiothreitol. Both forms had an apparently identical molecular weight of approximately 37,000/subunit and the same Km for fructose 1,6-bisphosphate of 2 microM. However, the Ki of the AMP inhibition of form A was 140 microM and of form B, 370 microM. With form B the same inhibition as with form A was reached by incubating the enzyme with dithiothreitol. The two forms of the enzyme differed in their total, as well as in their number of fast reacting thiol groups. Form A was the more reduced form, exhibiting 22.4 thiol groups/molecule, 2.5 of them fast reacting with 5,5'-dithiobis-(2-nitrobenzoic acid). Only 0.5 fast reacting groups and a total of 19.2 were found with form B. The fast reacting thiol groups disappeared when assayed in the presence of AMP. It is suggested that a redox reaction alters a site that influences the inhibitory action of AMP, so as to regulate the activity of fructose 1,6-bisphosphatase.     

The purification and characterization of a novel D(−)-specific carbamoylase enzyme from an Agrobacterium sp.

A carbamoylase enzyme was purified from a cell-free extract of Agrobacterium sp. with an overall yield of 81%. It was judged to be homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the native enzyme suggested that the active enzyme was present as a dimer, with a pI of 5.5. It was able to cleave a variety of N-carbamoyl substrates, but was strictly D(−) specific. It was found to have a K_m of 0.82 m and a V_max of 31 U mg⁻¹ for D(−) N-carbamoyl hydroxyphenylglycine in the presence of 10 m dithiothreitol. It showed no metal ion requirements but was inhibited by iodoacetic acid and iodoacetamide, both thiol reagents. The N-terminal amino acid sequence of the enzyme was elucidated.     

The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [¹²⁵I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn²⁺ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn²⁺- activated endopeptidase to be characterized.     

Aspergillus niger sulfhydryl oxidase

A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of a thiol protease in regenerating rat-liver nuclei. Partial purification and some properties

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525-531 (1077)]. The optimum pH was 5.5. 2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40 000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5 It was inhibited by thio reagents, E-64, leupeptin and hevy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones, alpha-N-Benzoylarginine-beta-naphthylamide and benzoylarginine amide were not hydrolyzed.     

Some properties of pig kidney-cortex aldehyde reductase.

Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.     

Purification and characterization of cathepsin B from monkey skeletal muscle

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.     

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.     

Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans.

1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.     

Purification and properties of a carboxylesterase from the liver of tiger shark (Galeocerdo cuvier).

A procedure is described for the purification of a carboxylesterase from shark liver, using a chloroform-acetone powder prepared from the liver as the starting material. The yield of purified enzyme is approximately 50 mg from 530 g of chloroform-acetone powder. The preparation is electrophoretically homogeneous. Active-site titrations with paraoxon gave an equivalent weight of approximately 83 000. The molecular weight, found from sedimentation equilibrium experiments, is approximately 80 000. There is no evidence of any association or dissociation of this species. The enzyme shows a marked preference for aryl esters over alkyl esters, in contrast to other carboxylesterases so far studied. The amino acid composition of the purified enzyme is reported.     

Purification and Properties of a Hydrogenase from Desulfovibrio vulgaris

The soluble hydrogenase of Desulfovibrio vulgaris was purified and some of its properties are described. The molecular weight was determined for the enzyme by sedimentation equilibrium (45,400) and amino acid analysis (44,800). The hydrogenase appears to be a loosely coiled molecule or to have a high axial ratio, which is reflected in an unusually low sedimentation coefficient (2.58S) and a low diffusion coefficient (D 5.85). The molecular weight of the hydrogenase (41,000), as calculated by the Svedberg equation, was in general agreement with the sedimentation equilibrium molecular weight. Amino acid analysis revealed the presence of six halfcystine residues per mole of enzyme and a total of 417 amino acid residues. The specificity of the hydrogenase and its capacity to reduce certain low potential dyes and cytochrome c(3) was studied. Metal analysis of the hydrogenase indicated the presence of 1 mole of ferrous iron per mole of enzyme.     

The thiol pool in human plasma: The central contribution of albumin to redox processes

The plasma compartment has particular features regarding the nature and concentration of low and high molecular weight thiols and oxidized derivatives. Plasma is relatively poor in thiol-based antioxidants; thiols are in lower concentrations than in cells and mostly oxidized. The different thiol-disulfide pairs are not in equilibrium and the steady-state concentrations of total thiols as well as reduced versus oxidized ratios are maintained by kinetic barriers, including the rates of reactions and transport processes. The single thiol of human serum albumin (HSA-SH) is the most abundant plasma thiol. It is an important target for oxidants and electrophiles due to its reactivity with a wide variety of species and its relatively high concentration. A relatively stable sulfenic (HSA-SO₃H) acid can be formed in albumin exposed to oxidants. Plasma increases in mixed disulfides (HSA-SSR) or in sulfinic (HSA-SO₂H) and sulfonic (HSA-SO₃H) acids are associated with different pathologies and may constitute biomarkers of the antioxidant role of the albumin thiol. In this work we provide a critical review of the plasma thiol pool with a focus on human serum albumin.     

Crystallization and properties of acylphosphatase from porcine skeletal muscle

A crystalline acylphosphatase has been obtained from porcine skeletal muscle. The purification procedure consists of extraction with water, phosphocellulose column chromatography, CM-cellulose column chromatography, and crystallization. The enzyme was homogeneous by polyacrylamide gel electrophoresis. A high yield (39%) of the pure enzyme was attained by the use of buffers containing 10 mM 2-mercaptoethanol to prevent dimerization of the enzyme in the purification process. Activity assay in the presence of bovine serum albumin showed a high specific activity of the enzyme (about 7,000 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate). The molecular weight was determined to be 11,100 by sedimentation equilibrium. The amino acid composition of the enzyme was determined. The amino-terminus of the enzyme was blocked and the carboxyl-terminal residue was tyrosine.     

Human alpha-N-acetylglucosaminidase. 1. Purification and properties.

alpha-N-Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. alpha-N-Acetylglucosaminidase is a glycoprrotein with an extensive charge heterogeneity. The molecular weight determined by gel filtration is 307000. Polycarylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 +/- 0.3 and KM and V values of 0.14-0.74 mM, and 1.04-3.68 mumol mg-1 min-1 for three aryl 2-acetamido-2-deoxy-alpha-D-glucosides and UDP-N-acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. --SH-protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65 degrees C and pH values below 5 inactivate the enzyme rapidly.     

Isolation of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Human Brain

Abstract: The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been isolated from an acetone powder of human subcortical white matter. The yield was about 11 mg from 28 g of powder and a specific activity of 213 unitdmg protein was obtained using 2',3'-cyclic CMP as the substrate. A major protein band of molecular weight approx. 96,000 was found by gel electrophoresis under nonreducing conditions. However, two distinct protein bands of molecular weight 46,000 ± 1400 and 48,000 ± 1400 were observed when the protein sample was reduced with 10 mM-dithiothreitol and subjected to electrophoresis in more restrictive 12-15% polyacrylamide-SDS gels. This molecular weight is lower than that previously reported for the bovine enzyme. Antibodies against the purified human enzyme have been raised in New Zealand white rabbits.     

Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.