Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Distribution of liver plasma membrane 5' nucleotidase as indicated by its reaction with anti-plasma membrane serum

Antiserum against mouse liver plasma membranes was used to investigate the properties and distribution of the surface membrane enzyme 5′ nucleotidase.The antiserum inhibited 5′ nucleotidase but had no effect on alkaline phosphodiesterase, nucleotide pyrophosphatase, or insulin-binding activity.5′ Nucleotidase was purified from mouse liver plasma membranes and the purified enzyme was shown to be inhibited by the antiserum. The membrane-bound and the purified enzyme were both inhibited in a noncompetitive manner.The reaction of the antiserum with 5′ nucleotidase activity of mouse liver plasma membrane “light” and “heavy” subfractions, and of rat liver and pig lymphocyte surface-membrane fractions was investigated. In each case the enzyme was inhibited by the antiserum.Since a protein must be partially exposed on the membrane surface in order to react with its antibody, the results are discussed in terms of the disposition of 5′ nucleotidase within the membrane.     

Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties

A proclotting enzyme associated with the hemolymph coagulation system of limulus (Tachypleus tridentatus) was highly purified from the hemocyte lysate. The first step of purification was performed by chromatography of the lysate on a pyrogen-free dextran sulfate-Sepharose CL-6B column, which was essential for separation of the proclotting enzyme from its activator, named factor B. The following steps consisted of column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-150, benzamidine-CH-Sepharose and Sephacryl S-300. Through these procedures, 1.4 mg of the purified material was obtained from 630 ml of the lysate and approximately 300-fold purification was achieved. The preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence and absence of 2-mercaptoethanol. The single-chain proclotting enzyme was a glycoprotein with an apparent molecular weight of 54,000, and no gamma-carboxyglutamic acid was detected. The proclotting enzyme was converted to its active form by purified factor B or by trypsin. The resulting clotting enzyme had a molecular weight of 54,000, consisting of a heavy chain of Mr = 31,000 and a light chain of Mr = 25,000. The serine active site of the clotting enzyme was found in the heavy chain. The chemical analyses of the isolated heavy and light chains indicated that the activation of the proclotting enzyme to its active form by factor B or trypsin is induced by a limited proteolysis, yielding two chains bridged by a disulfide linkage(s).     

A method for large scale purification of turnip peroxidase and its characterization

Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.     

Phospholipase C ofPseudomonas chlororaphis and its toxicity for insects

Phospholipase C (Ec 3.1.4.3) obtained fromPseudomonas chlororaphis was purified 70-fold and its activity toward various substrates was determined. The enzyme was found to have no toxic effect either on larvae ofGalleria mellonella or on the rabbit. The molecular weight of the enzyme was approximately 54,000, its pH optimum 7.5 and the isoelectric point at pH 6.3. The biochemical and toxogenic properties of the enzyme were compared with those ofBacillus cereus and ofClostridium perfringens phospholipases.     

Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme.

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed.     

Purification of turnip peroxidase and its kinetic properties

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA).     

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.     

A simple method for the recovery of active oligomeric enzyme from its diluted solutions

A method for the recovery of active enzyme from its diluted solutions is described. It includes batch sorption on DEAE-Sephadex A-50 which follows elution or chromatography of an enzyme on a DEAE-Sephadex A-50 column. All purified aminoacyl-tRNA synthetases retain high activity without degradation of the protein. The method is very fast and convenient.     

Immobilization of phosphorylase B and study of its enzymatic and immunological properties]

The properties of phosphorylase B (PhB) immobilized on an agar derivative were studied. It was shown that the enzyme activity makes up to 15-20% as compared to that of the soluble enzyme, the Km value for glucose-1-phosphate is increased 1.5-fold and the pH optimum remains unchanged, whereas the thermostability of enzyme shows a considerable increase. PhB immobilized on a highly activated sorbent completely losses its enzymatic activity but retains its antigenic properties and binds 1.6-2 mol antibodies (per monomer). Using immunosorbents, purified antibodies homogeneous during electrophoresis in polyacrylamide gel were isolated. The immunosorbent capacity is 500-800 mg of antibodies per 1 g of dry weight. The purified antibodies are characterized by a lower inhibitory power upon interaction with soluble PhB. The type of inhibition of both immobilized and soluble enzyme is similar. It is assumed that immobilization produces conformational changes only at the active site of enzyme, which is spatially separated from the antibody binding site.     

Porcine muscle prolyl endopeptidase and its endogenous substrates

Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.     

Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.     

Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties.

Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-adenosyl-L-methionine, oxamate, and reduced for ferredoxin or methylviologen. On the other hand, the purified enzyme was not reactivated. Either D-glyceraldehyde 3-phosphate or dihydroxyacetone phosphate strongly inhibited this enzyme. The inhibitory effects of these compounds were largely influenced by enzyme concentration. The inhibition of these triose phosphates in cooperation with the reactivating effect of ferredoxin and the fluctuations of both the enzyme and the triose phosphate levels may efficiently regulate the pyruvate formate-lyase activity in S. mutans in vivo.     

Changes in the activity of phosphogluconate dehydrogenase and its regulation in tobacco infected with PVY

The content and changes in the activity of phosphogluconate dehydrogenase were followed in leaf tissues of tobacco plants infected with the potato virus Y (PVY) in the acute phase of infection. The activity of the enzyme was higher in virus-infected tissues during the entire experimental period compared with the values found in healthy control plants in both crude homogenate and partially purified enzyme preparation. The courses of the activity curves of both the crude extract and partially purified enzyme preparations were consistent and correlated with the reproduction curve of the virus. These results suggest that increased activity of the enzyme was a result of its coarse regulation and indicate its immediate involvement in“de novo” biosynthesis of the virus via the oxidative pentose phosphate pathway.     

胆碱脱氢酶的底物保护作用

胆碱脱氢酶（ＣＤＨ）是线粒体电子传递酶系的一个重要组成,它位于线粒体内膜。膜固有的ＣＤＨ与用去垢剂从线粒体上增溶下来的酶在性质上有一定差异,本文研究了温度、ＳＤＳ对增溶ＣＤＨ的失活作用,发现底物胆碱的存在有明显的保护作用,说明底物诱导ＣＤＨ产主构象变化．     

An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin

We have purified to homogeneity the enzyme in the kidney cortex which accounts for the vast majority of matrix-degrading activity at neutral pH. The purified enzyme has an apparent molecular mass of 350 kD by gel filtration and of 85 kD on SDS-PAGE under reducing conditions; and it degrades laminin, type IV collagen and fibronectin. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by other proteinase inhibitors. The enzyme was not activated by organomercurials or by trypsin and was not inhibited by tissue inhibitors of metalloproteinases indicating that it is distinct from the other matrix- degrading metalloproteinases. Unexpectedly, the amino acid sequence of the NH2-terminal and two internal peptides of the enzyme showed complete homology to those alpha subunits of rat meprin, an enzyme previously shown to degrade azocasein and insulin B chain but not known to degrade extracellular matrix components. Immunoprecipitation studies, Western blot analyses and other biochemical properties of the purified enzyme confirm that the distinct matrix-degrading enzyme is indeed meprin. Our data also demonstrate that meprin is the major enzyme in the renal cortex capable of degrading components of the extracellular matrix. The demonstration of this hitherto unknown function of meprin suggests its potential role in renal pathophysiology.     

L-leucine and its analogue: specific inhibitors for S-benzyl-L-cysteine-p-nitroanilide-hydrolyzing enzyme in Escherichia coli B

An enzyme that catalyzes hydrolysis of S-benzyl-L-cysteine-p-nitroanilide was purified from E. coli B. The enzyme was a monomer with a molecular weight of 82,000. In addition to L-cysteinylglycine, the enzyme hydrolyzed various glycine-containing dipeptides most efficiently at pH 7.0. The enzyme required no metal ions for activity and was specifically inhibited by L-leucine and its analogue with free carboxyl group at the physiological concentrations.     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.