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1.
1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7.  相似文献   

2.
Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity.  相似文献   

3.
Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exo-large penicillinase (mol. wt, 305000; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.  相似文献   

4.
Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as 'D', all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and 'D' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are 2 mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and 'D' represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase 'D' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909-9912] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and 'D' have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to 'D'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.  相似文献   

5.
GH receptors (GHRs) have been shown by affinity cross-linking to be present in late pregnant mouse liver microsomes in three forms with cross-linked mol wts of 125,000, 62,000, and 56,000. The two lower mol wt forms of the receptor were partially purified by bovine GH-affinity chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate-solubilized extracts of late pregnant mouse hepatic microsomes. The GHRs were identified from the partially purified receptor preparation and isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated GHRs had mol wts of 40,700 and 37,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Enzymatic cleavage of N-linked glycosylation from the isolated GHRs reduced their apparent mol wts to 33,600 and 30,900, respectively. Sixteen of the amino-terminal 17 amino acid residues of the two isolated receptors were sequenced and determined to be identical. One amino acid residue in each of the proteins, at position 14, could not be identified. Rabbit polyclonal antiserum was produced against the isolated GHRs. The resulting antiserum precipitated the isolated 40,700 and 37,500 mol wt proteins as well as cross-linked mouse GHRs (including the high mol wt form of the receptor). However, the antiserum did not inhibit the binding of mouse GH to either membrane bound or 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate-solubilized GHRs.  相似文献   

6.
Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.  相似文献   

7.
Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.  相似文献   

8.
1. Fatty acid binding activity associated with a 14,000-15,000 mol. wt protein was observed in the cytosolic fraction of liver, duodenum, myocardium, adipose, pectoral and gastrocnemius muscles of chickens. 2. Polyclonal antisera prepared against chicken liver fatty acid binding protein affinity for only liver FABP and a 14,000 mol. wt fatty acid binding protein in the intestine. 3. A fatty acid binding protein was not detected in chicken plasma.  相似文献   

9.
The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.  相似文献   

10.
We synthesized six water-soluble polymeric complexes of sorbic acid with polyvinylpyrrolidone of different molecular weight (mol wt). As shown by infrared absorption spectrum analysis, the complexes were formed by hydrogen bonding. The complexes (SC1, with mol wt=10 kDa, SC2 with mol wt=25 kDa, SC3 with mol wt=30 kDa, SC4 with mol wt=40 kDa, SC5 with mol wt=90 kDa, and SC6 with mol wt=360 kDa) were characterized as low mol wt (SC1, SC2, and SC3) and high mol wt (SC4, SC5, and SC6). The antifungal potencies of the complexes were tested by the macrodilution susceptibility method against environmental and clinically important fungi. Sorbic acid as well as the complexes exhibited minimum inhibitory concentrations (MICs) lower than potassium sorbate against all the strains tested. MICs of SC1, SC2, and SC3 were shown to be 2- to 4-fold lower for yeast and 1.5- to 3-fold lower than those of sorbic acid for moulds, respectively. The MICs of SC4 and SC5 against both of the Candida species tested ranged from 500 to 800 microg/ml, whereas for SC6 and sorbic acid they were about 1 mg/ml. The potencies of the high mol wt complexes against moulds were decreased by increasing the mol wt. For both of the moulds tested, the MICs of SC4 were slightly lower than those of sorbate. The MICs of sorbic acid and SC5 were equal to 300 microg/ml and 500 microg/ml respectively for Aspergillus parasiticus and for Penicillum viridicatum. The susceptibility to SC6 of all of the hyphomycetes tested was higher than that to sorbic acid. The low mol wt complexes and the sorbic acid exhibited minimal fungicidal concentrations (MFCs) 2 and 3 times higher respectively than the MICs. Sorbic acid and SC3 at a concentration of 2.5 mg/ml in an in vitro time kill curve study of Candida tropicalis were shown to be fungistatic, whereas SC1 and SC2 were fungicidal at the same concentrations. For Aspergillus parasiticus sorbic acid at 2.5 mg/ml was fungistatic for a 24-h period, whereas SC1, SC2, and SC3 were fungicidal.  相似文献   

11.
1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.  相似文献   

12.
1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.  相似文献   

13.
Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.  相似文献   

14.
1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris(+) or triethanolamine(+). 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH.  相似文献   

15.
Mitochondrial extracts of dog, cat, rat and mouse liver contain two forms of alanine-glyoxylate aminotransferase (EC 2.6.1.44): one, designated isoenzyme 1, has mol.wt. approx. 80 000 and predominates in dog and cat liver; the other, designated isoenzyme 2, has mol.wt. approx. 175 000 and predominates in rat and mouse liver. In rat and mouse liver, isoenzyme 1 activity was increased by the injection in vivo of glucagon, but not isoenzyme 2 activity. Isoenzyme 1 was purified and characterized from liver mitochondrial extracts of the four species. Both rat and mouse enzyme preparations catalysed transamination between a number of L-amino acids and glyoxylate, and with L-alanine as amino donor the effective amino acceptors were glyoxylate, phenylpyruvate and hydroxypyruvate. In contrast, both dog and cat enzyme preparations were specific for L-alanine and L-serine with glyoxylate, and used glyoxylate and hydroxypyruvate as effective amino acceptors with L-alanine. Evidence that isoenzyme 1 is identical with serine-pyruvate aminotransferase (EC 2.6.1.51) was obtained. Isoenzyme 2 was partially purified from mitochondrial extracts of rat and mouse liver. Both enzyme preparations were specific for L-alanine and glyoxylate. On the basis of physical properties and substrate specificity, it was concluded that isoenzyme 2 is a separate enzyme. Some other properties of isoenzymes 1 and 2 are described.  相似文献   

16.
Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.  相似文献   

17.
The insulin-like growth factor-binding proteins IGFBP-1 and IGFBP-2 are low mol wt IGFBPs that are similar in structure. They are not glycosylated and have a homologous amino acid sequence, including the number and position of 18 cysteine residues and a carboxyl-terminal Arg-Gly-Asp sequence that can be recognized by cell adhesion receptors. The present study demonstrates that expression of mRNAs encoding the two BPs differs in some fetal rat tissues and in the livers of adult rats after hypophysectomy, fasting, or streptozotocin-induced diabetes. As determined by Northern blot hybridization using cDNA probes for rat IGFBP-2 or human IGFBP-1, both mRNAs are expressed at high levels in liver of 21-day gestation and 1-day-old rats and at lower levels in 21- and 65-day-old rat liver. Levels of both mRNAs are higher in liver than in other fetal rat tissues. The relative abundance of the two mRNAs in most fetal tissues is similar to that in liver, except that kidney and brain have 8-fold and more than 25-fold higher relative levels of IGFBP-2 mRNA, respectively. IGFBP-2 mRNA is about 10- to 20-fold increased after hypophysectomy or fasting, whereas IGFBP-1 mRNA is relatively unchanged. IGFBP-2 mRNA levels are decreased completely by refeeding fasted rats for 3 days, but only partially decreased by treatment of hypophysectomized rats with GH, cortisone acetate, T4, and testosterone for 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
1. Amino acid analyses are presented for a small molecular weight (mol. wt 11,000) protein (SMWP) obtained from the livers of normal chickens and chickens infected with erythroblastosis virus. It is resistant to acid denaturation and was isolated following acidification of liver homogenates and removal of haem and lipids. 2. Close homology exists between SMWP of fowl and human liver, and a protein of the same size obtained from chicken serum, (similar in size to human beta 2-microglobulin). 3. Human beta 2-microglobulin, virus core proteins and chicken prealbumin show a lower order relationship. 4. The amino acid residues of SMWP from healthy and erythroblastosis infected chicken livers are identical despite immunologic differences.  相似文献   

19.
A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).  相似文献   

20.
Haddock (Melanogrammus aeglefinus) is a gadoid fish species that deposits dietary lipid mainly in the liver. The fatty acid (FA) beta-oxidation activity of various tissues was evaluated in juvenile haddock fed graded levels of lipid. The catabolism of a radiolabelled FA, [1-(14)C]palmitoyl-CoA, through peroxisomal and mitochondrial beta-oxidation was determined in the liver, red and white muscle of juvenile haddock fed 12, 18 and 24% lipid in the diet. There was no significant increase in the mitochondrial or peroxisomal beta-oxidation activity in the tissues tested as the dietary lipid level increased from 12 to 24%. Peroxisomes accounted for 100% of the beta-oxidation observed in the liver, whereas mitochondrial beta-oxidation dominated in the red (91%) and white muscle (97%) of juvenile haddock. Of the tissues tested, red muscle possessed the highest specific activity for beta-oxidation expressed on a per mg protein or per g wet weight basis. However, white muscle, which forms over 50% of the body mass in gadoid fish was the most important tissue in juvenile haddock for overall FA catabolism. The total lipid and FA composition of these tissues were also determined. This study confirmed that the liver was the major lipid storage organ in haddock. The hepatosomatic index (HSI; 10.0-15.2%) and lipid (73.8-79.3% wet wt.) in the liver increased significantly as dietary lipid was increased from 12 to 24% lipid. There was no significant increase in the lipid composition of the white muscle (0.8% wet wt.), red muscle (1.9% wet wt.) or heart (2.5% wet wt.).  相似文献   

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