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.     

Carboxylesterases (EC 3.1.1). A comparison of some kinetic properties of horse, sheep, chicken, pig, and ox liver carboxylesterases.

A comparative study of the kinetic be,avior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificites towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis-Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simple scheme which accounts for both activation and inhibition is discussed. pH-k(cat) profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pK(a)S of 4.75 and 5.0, respectively.     

Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis.

The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.     

几种动物肝脏糖胺聚糖的醋酸纤维素薄膜电泳分析

采用酶解和离子交换色谱的方法,从兔、鸡、猪和羊肝组织中提取和纯化得到了糖胺聚糖（GAGs）.通过比较透明质酸（HA）、硫酸软骨素A（CS-A）、硫酸软骨素C（CS-C）、硫酸皮肤素（DS）、肝素（HP）、硫酸乙酰肝素（HS）等标准品在醋酸钡、醋酸锌、吡啶-甲酸等几种不同缓冲体系下的醋酸纤维素薄膜电泳行为,结合灰度积分建立了适合于微量GAGs定性和定量分析的电泳方法.将从不同动物肝脏组织中提取的GAGs运用该方法进行分析,发现 不同动物肝脏组织中,GAG含量和组成均有较大差异：羊肝中GAGs含量最高（0.52 mg/g 组织干粉）,种类也最丰富,含有HA、HS、DS和CS,其中HA所占比例最高;鸡肝中GAGs含量最少（0.18 mg/g组织干粉）,主要含有HA和DS;兔肝GAGs种类与猪肝相似,均含有HA、HS和DS,但HS是猪肝GAGs的主要成分,DS是兔肝GAGs的主要成分.     

Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.     

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.     

Characterization of intermediate-molecular-weight acid phosphatase from bovine kidney cortex

The distribution of acid phosphatases of intermediate molecular weight was determined in various mammalian tissues. The intermediate-molecular-weight acid phosphatases (designated P-II-1 and 2) comprised about 25% of the p-nitrophenyl phosphatase activity in the supernatant of bovine kidney cortex homogenate. The P-II-1 and 2 purified 2,000 fold showed the pI values of 5.9 and 5.7, respectively, on isoelectric focusing. Apparent molecular weights of both P-II-1 and 2 were estimated to be 42,000 by Sephadex G-100 gel filtration and 44,000 by SDS-polyacrylamide disc gel electrophoresis. Both the enzymes catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for the phosphoproteins phosphoserine and o-phosphocholine. The enzymes showed a high activity on pyridoxal phosphate, beta-glycerophosphate, and 2'-AMP. The optimum activity pH was near 5 with p-nitrophenyl phosphate, but was shifted to the neutral range when pyridoxal phosphate was the substrate. The cations Hg2+ and Ag+ had a marked inhibitory effect. Neither enzyme was inhibited significantly by L-(+)-tartrate or pCMB. The two other types of acid phosphatases, the high-molecular-weight (designated P-I) and low-molecular-weight (designated P-III), were also purified to homogeneity from bovine kidney cortex, and were compared with P-II from several aspects including substrate specificity and susceptibility to various compounds.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.     

THE REGIONAL DISTRIBUTION, AGE DEPENDENT VARIATION AND SPECIES DIFFERENCES OF BRAIN ARYLSULPHATASES

Abstract— The relative proportions of arylsulphatase A and B were determined by the method of B aum , D odgson and S pencer (1959) in brains of various animal species and it was found that there was a considerable variation in the concentration of these two enzymes.
Arylsulphatase A and B of various animal species including rat, man, monkey, sheep and chicken were partially separated using zinc acetate fractionation procedure and gel electrophoresis. The chicken brain arylsulphatase A had a similar electrophoretic mobility to that of arylsulphatase B of other species. Further, chicken brain arylsulphatase A precipitated at a zinc acetate concentration of 0005 M, a condition under which arylsulphatase B from the brain of other species precipitated.
Kinetic properties such as K _m value and inhibitory effect of sulphite and phosphate ions indicated that chicken brain arylsulphatase A was similar to arylsulphatase A of other species.
The results on regional distribution of arylsulphatase A and B activities in monkey brain and in developing rat brain suggest a relationship between arylsulphatase A and sulphatides and arylsulphatase B and mucopolysaccharides.     

A novel acid phosphatase excreted by Penicillium funiculosum that hydrolyzes both phosphodiesters and phosphomonoesters with aryl leaving groups

An acid phosphatase has been purified from the culture broth of Penicillium funiculosum by procedures including SP-Sephadex column chromatography and Sephacryl S-200 gel filtration. The phosphatase appears to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes both phosphodiesters and phosphomonoesters, but only those with aryl leaving groups. At the early phase of degradation of bis-p-nitrophenyl phosphate by the enzyme, inorganic phosphate and the intermediary product, p-nitrophenyl phosphate, are liberated at approximately the same rate. This indicates that the intermediary phosphomonoester produced on the enzyme is further hydrolyzed in situ or dissociates into the medium at approximately the same probability.     

Alkaline phosphatase from Echinococcus multilocularis: purification and characterization.

1. Kinetic and physical parameters of purified alkaline phosphatase from Echinococcus multilocularis metacestodes, livers of infected gerbils and control animals were determined. 2. Km value for p-nitrophenyl phosphate was about 0.05 +/- 0.02 mM for the three enzymes. 3. Vmax values were 357 +/- 67 nmol/min/mg proteins for metacestode enzyme, and 6.7 +/- 1.1 and 6.7 +/- 0.8 nmol/min/mg proteins for liver enzyme of infected and control animals, respectively. 4. Mr and pI were different for the parasite and hepatic enzyme. 5. The parasite enzyme was less sensitive to the elevation of temperature than hepatic enzyme. 6. The isatin inhibition was a competitive inhibition type for parasite and uncompetitive type for host liver enzyme.     

Purification and properties of poly(ADPribose) synthetase from sheep testis

Poly(ADPribose) synthetase has been purified to apparent homogeneity from sheep testis by a simple procedure using three chromatographic steps (DNA-agarose, blue Sephadex G-150 and phosphocellulose P11). A concentrated enzyme preparation, 3.5 mg, with a specific activity of 1265 nmol/min per mg was obtained from 250 g of tissue. DNA was absolutely required for enzyme activity. The half-maximal activation occurred at the concentrations of 11 micrograms/ml for highly polymerized calf thymus DNA and 2 micrograms/ml for sonicated calf thymus DNA. The Km for NAD was 57 microM. The molecular weight was 120 000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated that the main amino acid species of sheep testis enzyme were very similar to those of enzymes from other sources.     

Cellular retinol-binding protein from rat liver. Purification and characterization

Cellular retinol-binding protein has been purified to homogeneity from rat liver. The procedures utilized in the purification included acid precipitation, gel filtration on Sephadex G-75 and G-50, and chromatography on DEAE-cellulose. The binding protein was purified approximately 3,500-fold, based on total soluble liver protein. The protein is a single polypeptide chain with a molecular weight of 14,600 based on information obtained by the techniques of sedimentation equilibrium analysis, gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinol with high affinity; the appparent dissociation constant was determined by fluorometric titration to be 1.6 X 10(-8) M. Retinol bound to the protein has an absorption spectrum (lambdamax, 350 nm) considerably altered from the spectrum of retinol in ethanol (lambdamax, 325 nm).     

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.