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1.
Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle 总被引:2,自引:0,他引:2
D Mantle B Lauffart J R McDermott A M Kidd R J Pennington 《European journal of biochemistry》1985,147(2):307-312
Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides. 相似文献
2.
Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber. 总被引:2,自引:0,他引:2
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1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed. 相似文献
3.
Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose. 相似文献
4.
Summary Proteases were the principal secretory proteins of Clostridium sporogenes and were optimally produced after active growth at 37° C. Glucose, ammonia and peptides repressed protease production. Protease formation was maximal in cultures grown at pH 6.5, but proteolytic activity exhibited a pH optimum of 7.0–8.0. Protease activity in culture filtrates was stimulated by divalent metal ions (Ca2+, Mn2+ and Co2+) and was strongly inhibited by ethylene diaminetetraacetate (EDTA) and thimerosal. Non-denaturing polyacrylamide gel electrophoresis and HPLC gel filtration demonstrated the presence of six major proteases of low molecular mass (approx. 15–35 kDa). The enzymes were partially purified from non-denaturing gels. Each hydrolysed azocoll and azocasein, but differed in their activity against a range of native collagen substrates. All six enzymes degraded human placental collagen (Type IV) but only one had a broad substrate specificity, being able to hydrolyse the more recalcitrant collagens (Types I, II and III). Experiments with individual proteases showed that their activities were strongly inhibited (40–85%) by 5 mM EDTA, indicating that they were metalloproteases. The enzymes exhibited different susceptibilities to inhibition by either 3 mM phenylmethylsulphonyl-fluoride (PMSF), 5 mM thimerosal, or 10 mM cysteine, which respectively inhibit serine, thiol and metalloproteases. 相似文献
5.
I.P. Belyauskaite V.J. Palubinskas O.E. Anchenko V.S. Vesa A.A. Glemzha 《Enzyme and microbial technology》1980,2(1):37-44
A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ~35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca2+, Mg2+, Cu2+, Co2+) on proteolytic activities of these enzymes studied. 相似文献
6.
《Process Biochemistry》1999,34(5):441-449
Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca2+ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The Km values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P1 position. 相似文献
7.
Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues. 总被引:3,自引:0,他引:3
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Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin. 相似文献
8.
Purification and Characterization of a Neuropeptide-Degrading Aminopeptidase from Human Brain 总被引:3,自引:2,他引:1
The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis. 相似文献
9.
Two new esterases (JEA and JEB) and a lipase (JL) were extracted from the seeds of Jatropha curas L. Lipase activity was only found during germination of the seeds and increased to a maximum after 4 days of germination. All enzymes were found to be most active in the alkaline range at around pH 8 and the purified (fractionated precipitation with ethanol and gel filtration) esterases were very stable at high temperatures. The molecular weight (SDS-PAGE) of both esterases was determined to be 21.6-23.5 kDa (JEA) and 30.2 kDa (JEB) and the isoelectric point was 5.7-6.1 for esterase JEA and 9.0 for esterase JEB. Most ions caused a negative influence on the activity of both esterases. Using p-nitrophenyl butyrate as a substrate JEA showed a K(m) of 0.02 mM and a v(max) of 0.26 micromol mg(-1) min(-1). Under the same conditions JEB showed a K(m) of 0.07 mM and a v(max) of 0.24 micromol mg(-1) min(-1). Both esterases hydrolyzed tributyrin, nitrophenyl esters up to a chain length of =C4 and naphtylesters up to a chain length =C6. In transesterification reactions, JL was found to be most active at very low water activities (0.2) and in high water activities, the lipase hydrolysed triglycerides into conversions above 80%. The lipase hydrolysed both short chain and long chain triglycerides at about the same rate but was inactive on alpha-methylbenzyl acetate. JL is a potentially useful biocatalyst in the hydrolysis of triglycerides in organic solvents. 相似文献
10.
Two undecapeptide substance P (SP) analogues, Spantide I and Spantide II, were tested for their capacity to block the contractile effect of SP on the guinea pig isolated taenia coli and the contractile effect of electrical stimulation of the rabbit isolated (and atropinized) iris sphincter, and for their capacity to mobilize histamine from rat isolated peritoneal mast cells. Spantide I and Spantide II have one feature in common, namely D-tryptophan in positions 7 and 9. Spantide I: D-Arg, Pro2, Lys3, Pro4, Gln5, Gln6, D-Trp7, Phe8, D-Trp9, Leu10, Leu11-NH2. Spantide II: D-NicLys1, Pro2, 3-Pal3, Pro4, D-Cl2Phe5, Asn6, D-Trp7, Phe8, D-Trp9, Leu10, Nle11-NH2. Both Spantide I and II were found to be competitive antagonists to SP on the taenia coli and to be capable of blocking the electrically induced non-cholinergic contraction of the iris sphincter. Spantide II had higher pA2 value (taenia coli) than Spantide I, 7.7 versus 7.0, and higher pIC50 value (blockade of tachykinin-mediated neurotransmission in iris sphincter), 6.0 versus 5.1. Both Spantide I and II mobilized histamine from rat peritoneal mast cells but Spantide II was less effective. Spantide I and II were tested for antagonistic specificity. Both blocked contractions of the taenia induced by SP and neurokinin A. In the concentration used, Spantide II in addition blocked the response to neurokinin B. The contractions induced by carbachol, 5-hydroxytryptamine, histamine and prostaglandins (F2 alpha and E1) were not affected; the contractile response to bombesin was inhibited by Spantide I but not by Spantide II. 相似文献
11.
López V Alarcón R Orellana MS Enríquez P Uribe E Martínez J Carvajal N 《The FEBS journal》2005,272(17):4540-4548
To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II. 相似文献
12.
Silva RA Yasui SC Kubelka J Formaggio F Crisma M Toniolo C Keiderling TA 《Biopolymers》2002,65(4):229-243
Model peptides based on -(Aib-Ala)(n)-, and (Aib)(n)-Leu-(Aib)(2) sequences, which have varying amounts of 3(10)-helical character, were studied by use of vibrational and electronic circular dichroism (VCD and ECD) and Fourier transform infrared (FTIR) absorption spectroscopies to test the correlation of spectral response and conformation. The data indicate that these peptides, starting from a length of about four to six residues, predominantly adopt a 3(10)-helical conformation at room temperature. The longest model peptides, depending on the series, may evidence some alpha-helical contribution to the spectra, while the shorter ones, with less than six residues, have much less order. The IR absorption spectra (as supported by theory) showed only small frequency changes between 3(10)- and alpha-helices. By contrast, solvent effects are a source of much bigger perturbations. The ECD results show that the intensity ratio for the approximately 222-nm to approximately 208-nm bands, while useful for distinguishing between these two helical types in some sequences, may have a narrower range of application than VCD. However, the VCD data presented here continue to support the proposed discrimination between alpha- and 3(10)-helices based on qualitative amide I and II bandshape differences. The present study shows the intensities of the 3(10)-helical amide I (peak-to-peak) to its amide II VCD to be of the same order and useful for discriminating them from alpha-helices, whose amide I dominates the amide II in intensity. This qualitative result is experimentally independent of the amount of alphaMe-substituted residues in the sequence. These experimental VCD results are consistent in detail with theoretical spectral simulations for Ac-(Ala)(8)-NH(2), Ac-(Aib-Ala)(4)-NH(2), and Ac-(Aib)(8)-NH(2) in 3(10)- and alpha-helical conformations. 相似文献
13.
Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag+ and iodoacetic acid. 相似文献
14.
The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar. 相似文献
15.
Bovine skeletal muscle contains small amounts of at least six heat- and acid-stable RNA-degrading enzymes. Our results are the first evidence for multiple ribonucleases in skeletal muscle. Three of these have been highly purified, and each has been shown to be a pyrimidine-specific endoribonuclease by use of a rapid sequencing technique employing gel electrophoresis. However, synthetic co-polymers containing adenylate or guanylate residues in addition to pyrimidine residues are hydrolysed at higher rates than are the pyrimidine homopolymers. With 0.63 mM yeast RNA as substrate, all three enzymes (ribonucleases I, II and III) are optimally active in alkaline solution (pH 7.5-8.5) containing 0.05-0.15 M univalent salts, do not require bivalent cations, and have molecular weights of 13 000-20 000. The properties of muscle ribonuclease I are very similar to those of bovine pancreatic ribonuclease A. Muscle ribonucleases II and III have characteristics similar to those of ribonucleases found in various other bovine tissues. In common with all previously studied pyrimidine-specific endoribonucleases, the bovine muscle ribonucleases are inhibited by such purine homopolynucleotides as polyadenylate. Furthermore, polyamines, present in low concentrations, can reverse or regulate the amount of inhibition of enzyme activity. 相似文献
16.
Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function 总被引:4,自引:0,他引:4
Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis. 相似文献
17.
Synthesis and properties of 7-hydroxymethotrexate polyglutamyl derivatives in Ehrlich ascites tumor cells in vitro 总被引:2,自引:0,他引:2
G Fabre I Fabre L H Matherly J P Cano I D Goldman 《The Journal of biological chemistry》1984,259(8):5066-5072
The synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-4-NH2-10-CH3-pteroyl-glutamic acid (PteGlu1] was evaluated by direct hydroxylation of the tetraglutamyl derivative of methotrexate (4-NH2-10-CH3-PteGlu4) by a cell-free preparation of rabbit liver aldehyde oxidase and by polyglutamylation of 7-OH-methotrexate in Ehrlich ascites tumor cells in vitro. The polyglutamyl derivatives of 7-OH-methotrexate rapidly accumulate in cells to the 7-OH-4-NH2-10-CH3-PteGlu4. While 7-OH-methotrexate monoglutamate does not bind to dihydrofolate reductase, 7-OH-4-NH2-10-CH3-PteGlu4 does bind to the enzyme as established by gel filtration analysis of cell extracts and by use of purified dihydrofolate reductase from Ehrlich cells. Within cells, the rate of formation of 7-OH-methotrexate polyglutamyl derivatives exceeds that for methotrexate by a factor of 2.7 at comparable free monoglutamyl substrate levels, suggesting that 7-OH-methotrexate may be a better substrate than methotrexate for the folylpolyglutamate synthetase. 7-OH-methotrexate slows the rate of methotrexate polyglutamylation in cells, a consequence of the inhibition of methotrexate transport with reduced methotrexate substrate available for polyglutamylation. When 7-OH-methotrexate polyglutamyl derivatives were accumulated inside the cells following which extracellular 7-OH-methotrexate was removed, the monoglutamate, and to a lesser extent the diglutamate, exited the cells whereas the majority of the longer polyglutamyl derivatives were retained and continued to be metabolized to higher forms. These studies suggest that 7-OH-methotrexate and its polyglutamyl derivatives may play a role in modulating methotrexate action, either by their own inhibitory effects on folate-dependent enzymes or by their effects on methotrexate transport and metabolism within cells. 相似文献
18.
A method of isolation of three different, partially purified deoxyribonucleases from the cells of Brevibacterium ammoniagenes is descrirbed. The enzyme preparations were activated by various bivalent metal ions: 50mM MgCl2 (I), 5 mM CaCl2+5 mM MgCl2 (II), 10 mM CaCl2 (III), and had different pH optima -- 8.8 (I), 7.2 (II) and 8.2 (III). In the isolated nuclei of rat brain the first and third fractions split chromatin at the internucleosomal sites with a formation of nucleosomes -- structural subunits of chromatin. The second fraction exhibited no structural specificity for chromatin. A possible use of the enzymes for the analysis of chromatin structure is discussed. 相似文献
19.
J. R. McDermott D. Mantle B. Lauffart A. M. Gibson J. A. Biggins 《Journal of neurochemistry》1988,50(1):176-182
Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate). 相似文献
20.
Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors. 总被引:5,自引:0,他引:5
Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control. 相似文献