固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

Markers of follicle function in Belclare-cross ewes differing widely in ovulation rate.

High prolificacy due to a gene that has a large effect on ovulation rate has been noted in Booroola and Inverdale ewes. High prolificacy in the Belclare breed (a composite developed from stocks selected for very large litter size or high ovulation rate) may be related to the segregation of two genes. The aims of this study were (i) to compare the morphological and functional features of ovulatory follicles from carriers (which could only be heterozygous for the genes of interest) and non-carriers, and (ii) to identify markers of the Belclare genes among secreted or cellular ovarian proteins. Belclare carrier ewes had more ovulatory follicles (4.9 +/- 0.4) than did non-carrier ewes (2.0 +/- 0.2) (P < 0.001). Ovulatory follicles from carriers were also smaller (4.4 +/- 0.1 mm versus 5.7 +/- 0.2 mm, P < 0.001) and contained a significantly reduced number of granulosa cells (P < 0.001). However, the proportion of proliferating granulosa cells in ovulatory follicles was similar in both groups. The in vitro secretion of steroids per follicle was only marginally lower in follicles from Belclare carriers compared with non-carriers. Furthermore, similar concentrations of steroidogenic enzymes were present in both groups, indicating that steroidogenic potential per granulosa cell is similar between carriers and non-carriers. Possible markers of the Belclare genes were identified among cellular proteins of follicular walls by two-dimensional PAGE and image analysis. Two spots at 78 and 49 kDa were always absent in samples from non-carriers. When secreted proteins in follicles from carriers were compared with those from non-carriers, two spots at 53 and 41 kDa were restricted to samples from carriers and three spots at 97, 91 and 45 kDa were unique to samples from non-carriers. Interestingly, the spot at 91 kDa is also affected by the Booroola gene.     

In Vitro Stability and Inactivation of Peptide Hydrolases Extracted from Phaseolus vulgaris L.

Endopeptidase activity against azocasein had a higher temperatureoptimum (50°C) in leaf extracts than in cotyledon extracts(37°C). The temperature optima for aminopeptidase (46°C)and for carboxypeptidase (53°C) were similar in leaf andcotyledon extracts. The endopeptidase activity showed an excellentstability in crude extracts from leaves even at 37°C, whilethe endopeptidase in cotyledon extracts was less stable. Carboxypeptidasewas very stable in both leaf and cotyledon extracts. Aminopeptidasewas the least stable of the enzymes investigated and its inactivationrate depended on the source of the extract. A moderate stabilitywas observed in extracts of leaves or of ungerminated seeds,but this enzyme was rapidly inactivated in cotyledon extractsat pH 5.4. At pH 7.5 aminopeptidase remained active longer thanat pH 5.4. From experiments with mixed extracts it could beconcluded that in cotyledons an aminopeptidase inactivatingfactor was formed during germination. This factor was heat sensitive,excluded by Sephadex G-25, precipitated by 75% ammonium sulfateand inhibited by tosyl-L-lysine chloromethyl ketone. These datasuggest that the factor is a protein and considering the similarproperties it appears possible that it is the endopeptidaseformed during germination. (Received May 15, 1981; Accepted July 18, 1981)     

Pear polygalacturonases

The polygalacturonase activity in extracts of ripe D'Anjou pears is due to two enzymes, which can be resolved by chromatography on Sephadex G-100. One     

Diphenoloxidases in various forms of myopathy which are transmitted by different genetic mechanisms

Summary In a previous article (Demos et al. 1981), we reported a significant and specific reduction of the activity index (AI) of the diphenoloxidases (DPox) in patients and heterozygotes with progressive Duchenne muscular dystrophy (DMD), which is transmitted genetically by female subjects by a sex-linked recessive mechanism (SLR). This same anomaly was detected in patients suffering from other types of dystrophy: Becker, limbgirdle, fascio-scapulo-humeral, and in heterozygotes, of either sex in diseases transmitted by an obviously recessive autosomic mechanism. These anomalies were detected using blood spots collected on absorbent paper and stored at 4°C for differnt periods. They were of the same type as had previously been detected using blood platelets (Demos 1973).     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.     

Adenylosuccinate synthetase and adenylosuccinate lyase from plant tissues

1. Enzymes that convert IMP into adenylosuccinate (adenylosuccinate synthetase) and adenylosuccinate into AMP (adenylosuccinate lyase) were isolated from wheat germ and pea seeds and their properties are described. 2. These enzymes were purified approx. 200-fold from wheat-germ extracts. 3. A heat treatment provided adenylosuccinate lyase free of adenylosuccinate synthetase but the behaviour of the two enzymes was almost identical in a number of fractionation procedures. The two activities were finally separated by filtration on Sephadex G-100. 4. The identification of these enzymes in plant tissues is discussed in relation to the pathway of purine synthesis.     

Isolation of aldehyde oxidase (EC 1.2.3.1) from potato extracts by preparative page

A method is proposed for the isolation (and purification) of enzymes, with retention of their activity, from solutions or gels of preparative PAGE runs. It is based on the inclusion of Sephadex G-25 as a supporting medium for a collector buffer in otherwise normal disc-PAGE gels. The collector buffer has a lower pH and higher concentration than the stacking gel buffer. This makes the proteins concentrate in a very narrow, slowly moving band in the Sephadex on electrophoresis, and makes their recovery easy. The method is illustrated by the isolation of aldehyde oxidase from potato extracts (which was unsuccessful by classical methods), and of one isoenzyme from commercial lipoxygenase after preparative PAGE. Recovery of chicken egg albumin after PAGE was over 90%.     

Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.     

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach

In order to identify target antigens of anti-endothelial cell (anti-EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2-DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 microg/mL. Targeted spots were identified by MS. The HUVEC proteome was composed of 884 protein spots. Among these, 61 +/- 25.8 (mean +/- SD) spots were recognized by serum IgG from healthy individuals, with marked differences from one individual to another. Among these spots, 11 were recognized by serum IgG from all healthy individuals tested. These spots corresponded to six different proteins with several spots corresponding to different isoforms of the same protein. Target antigens were: cytoskeletal proteins (beta-actin, alpha-tubulin, and vimentin); glycolytic enzymes (glucose-3-phosphate-deshydrogenase and alpha-enolase); and prolyl-4-hydroxylase beta subunit, a member of the disulfide isomerase family. This study shows that the repertoire of IgG AECA is heterogeneous among healthy individuals. IgG from all of the healthy individuals tested recognized a restricted set of highly conserved ubiquitous proteins playing key roles in cell biology and maintenance of homeostasis.     

Regulation of two aspartokinases in Bacillus subtilis

When grown on minimal glucose medium, transformable Bacillus subtilis strains contained two distinct aspartokinases (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4). One of these enzymes was inhibited by l-lysine (Lys), whereas the other was insensitive to inhibition but was activated by l-leucine. None of the other amino acids tested had any effect, and the addition of l-threonine did not enhance the inhibition by Lys, in contrast to the concerted inhibition observed for other bacilli. At the end of exponential growth, the Lys-sensitive aspartokinase activity decreased, whereas the Lys-insensitive activity remained relatively constant throughout the stationary phase. The two activities were separated by (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Growth in the presence of Lys reduced the specific activity of aspartokinase by about 50% and eliminated the inhibition by Lys. In extracts of these cells, only Lys-insensitive activity was found upon (NH(4))(2)SO(4) fractionation and Sephadex G-200 chromatography. Lys apparently repressed the synthesis of the Lys-sensitive enzyme.     

Isolation and characterization of NAD(P)H-dehydrogenases from seeds of the castor bean

Two pyridine nucleotide dehydrogenases have been isolated from castor bean seed extracts by a combination of ion exchange chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G-200. The enzymes were designated D-I and D-II according to their elution position on DEAE-Sepharose. Both enzymes D-I and D-II are globular proteins which have MWs of 66 000 and 60 000, respectively. Dehydrogenation is observed with both NADH and NADPH as electron donors, while the electron acceptor specificity demonstrates that the enzymes are probably NAD(P)H: quinone oxidoreductases. Successful coupling of dehydrogenase activity with that of peroxidase indicates a possible role of the enzymes in seed germination.     

Isolation and partial characterization of rat elastolytic enzymes from various cells and tissues

Different elastolytic enzymes were isolated from rat aorta and platelets, as well as from granulocyte and pancreatic extracts. The active fractions were purified to electrophoretic apparent homogeneity by precipitation with ammonium sulfate, sequential batch fractionation on DEAE-Sephadex A-50, and finally by isoelectric focusing (IF) on Sephadex G-75 Superfine. The molecular weight and the isoelectric point of the isolated enzymes were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by analytical IF, respectively. All the enzymes have elastolytic activity as well as activity toward Suc-(Ala)3-NA. The inhibition profile of the different isolated enzymes toward various inhibitors indicates that aortic, pancreatic, and granulocyte enzymes all belong to the group of serine proteinases, unlike the platelet elastase which is a metalloproteinase.     

Purification of Thiol-disulfide Interchange Enzyme from Candida claussenii

Thiol-disulfide interchange enzyme which catalyzes the thiol-disulfide interchange was purified from cell-free extracts of Candida claussenii by acid treatment, ammonium sulfate fractionation, aqueous polymer two phase method (Dextran-PEG system), CM-Sephadex column chromatography, Sephadex G–100 and Sephadex G–200 gel filtrations. More than four active fractions were obtained on CM-Sephadex column. Further purification steps from one of these fractions resulted in two purified enzyme preparations D–l–1 and D–2 of which the increase in specific activities was 8150- and 8450-folds respectively, over the crude extract. Both purified enzymes were homogeneous in ultracentrifugal analysis.     

Radioimmunoassays for prostaglandins. I. Technical validation of prostaglandin F2α measurements in human plasma using Sephadex G-25 gelfiltration

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Radioimmunoassays for prostaglandins. II. Measurement of prostaglandin E2 and the 13,14-dihydro-15-keto metabolites of the E and F series. Description of a reliable technique with a universal applicability

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C_18:0 and C_16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.

The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K_m values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

     

Biosynthesis of ethylene. Enzymes involved in its formation from methional

1. Two enzymes were shown to be necessary for the production of ethylene from methional; they were separated from extracts of cauliflower florets by fractionation on Sephadex and other methods. 2. The first enzyme, generating hydrogen peroxide, appears to be similar to the fungal glucose oxidase, for like the latter it is highly specific for its substrate d-glucose. 3. The second enzyme, in the presence of cofactors and peroxide generated by the first enzyme, cleaves methional to ethylene. 4. It was also found that hydrogen peroxide in these reactions may be replaced by hydroperoxide generated from linolenic acid by lipoxidase enzymes. 5. Dihydroxyphenols were shown to have a marked inhibitory effect on these reactions and to account for the initial phase of low activity that is always observed in aqueous extracts prepared from the floret tissue.