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1.
HELEN G. HANSMA† 《The Journal of eukaryotic microbiology》1975,22(2):257-259
The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethanol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (∼250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors. 相似文献
2.
We have studied four strains of Tetrahymena thermophila, each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3H]ethanolamine or [3H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide electrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-linked glycosyl-phosphatidylinositol anchors. 相似文献
3.
We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [3 H]ethanolamine or [3 H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [3 H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [3 H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors. 相似文献
4.
Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia. 总被引:15,自引:9,他引:6
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A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei. 相似文献
5.
Three Ca-dependent proteases have been identified in rat brain and skeletal muscle using ion exchange, gel filtration, and substrate affinity chromatography. A high degree of homology exists among three enzymes from different sources. Both the high molecular weight protease (154,000) and lower molecular weight protease (96,000) show high affinity for calcium while the third protease (76,000) had low affinity for calcium. Transformation among the three enzymes was calcium-induced and the process was unidirectional, generating a lower molecular weight form with decreased affinity for calcium. The protease with low affinity for calcium was susceptible to calcium-induced inactivation by autocatalysis. Immunologically the three proteases were equivalent, if not identical, and the brain and muscle proteases cross-react. All three proteases degraded neurofilament proteins; however, the protease with low affinity for calcium had 3 to 6 times higher specific activity. It is suggested that the high molecular weight enzyme (154,000) may be the native form of the Ca-dependent protease present in vivo. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2020-2023
Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein. 相似文献
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8.
The relationship between a large molecular weight (9S) and a small molecular weight (4.5S, 60,000 molecular weight) species of phytochrome was examined to determine if the larger species was an aggregate of the smaller. Alterations of pH, salt concentration, or phytochrome concentration did not cause any observable formation of the large form from the small form. However, in partially purified phytochrome extracts from Secale cereale L. and Avena sativa L., the large form was converted to the small form over time at 4 C in the dark. This breakdown was inhibitable by the protease inhibitor phenylmethanesulfonyl fluoride. When highly purified large molecular weight rye phytochrome was incubated with a neutral protease isolated from etiolated oat shoots, the large phytochrome was converted to the small form without qualitative visible absorbancy changes. The effect of the oat protease could be mimicked by a wide variety of commercial endopeptidases, including trypsin. Examination of the trypsin-induced breakdown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that as the size of the photoreversible unit changes from large to small, the size of its constituent polypeptide chains is reduced from 120,000 to 62,000 molecular weight. These experiments provide evidence that the endogenous breakdown observed in extracts is a result of contaminant protease and, consequently, that the small molecular weight species of phytochrome is an artifact due to proteolysis. 相似文献
9.
羊毛防毡缩用蛋白酶的化学修饰 总被引:1,自引:0,他引:1
为减少防毡缩整理中蛋白酶对羊毛纤维主体结构的破坏作用,分别研究了戊二醛、微生物谷氨酰胺转氨酶(MTG)和水溶性碳二亚胺(EDC)对蛋白酶Savinase 16L的化学修饰,以期达到增大蛋白酶分子量,从而将水解作用限制在纤维表面的目的。主要通过体积排阻色谱、SDS-PAGE谱图以及荧光光谱研究修饰酶的分子量和结构变化。结果表明,戊二醛不能对蛋白酶分子进行有效修饰;MTG会被蛋白酶水解,无法催化酶分子间发生共价交联;而碳二亚胺既可以使蛋白酶分子间发生交联,又能将含有伯胺基的大分子修饰剂偶联到酶分子上。 相似文献
10.
We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles in vitro and found that hFF1 contains a factor of high molecular weight (Mr greater than 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr less than 1000) which stimulated PG synthase activity. If this is true of ovarian follicles in vivo, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation. 相似文献
11.
We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles
and found that hFF contains a factor of high molecular weight (Mr > 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr < 1000) which stimulated PG synthase activity. If this is true of ovarian follicles
, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation. 相似文献
12.
Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease 总被引:2,自引:0,他引:2
Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon. 相似文献
13.
Purification and characterization of a calcium-activated neutral protease from monkey cardiac muscle 总被引:3,自引:0,他引:3
A calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+ and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110,000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74,000 and 28,000 and the more acidic one (form II CANP) contained components with molecular weights of 74,000 and 26,000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myofibrils by this protease resulted in the production of a component with a molecular weight of 30,000 as well as various other higher and lower molecular weight peptide fragments. 相似文献
14.
15.
Ghosh A Chakrabarti K Chattopadhyay D 《Journal of industrial microbiology & biotechnology》2008,35(8):825-834
Biotreatment of feather wastes and utilization of the degraded products in feed and foodstuffs has been a challenge. In the present study, we have demonstrated the degradation of feather waste by Bacillus cereus DCUW strain isolated during a functional screening based microbial diversity study on East Calcutta Wetland Area. A high molecular weight keratinolytic protease from feather degrading DCUW strain was purified and characterized. Moreover, utilization of degraded products during feather hydrolysis was developed and demonstrated. The purified keratinolytic protease was found to show pH and temperature optima of 8.5 and 50 degrees C, respectively. PMSF was found to inhibit the enzyme completely. The purified enzyme showed molecular weight of 80 kDa (from SDS-PAGE). The protease was found to have broad range substrate specificities that include keratin, casein, collagen, fibrin, BAPNA and gelatin. The protease was identified as minor extracellular protease (Vpr) by RT-PCR and northern blotting techniques. This is the first report describing the characterization of minor extracellular protease (Vpr) and its involvement in feather degradation in B. cereus group of organisms. 相似文献
16.
Female rabbit liver cytosol contains a receptor-modifying activity that converts the 250,000 estrogen receptor of liver and uterine cytosol to a 37,000 form. There is an age-dependent increase in this receptor-active protease and in the general protease activity of rabbit liver cytosol, measured with [14C]casein. Sephacryl S-200 chromatography of liver cytosol shows that in the young animal (5 weeks old) the major receptor-modifying activity elutes near the void volume, while in the older animal (13 weeks old) activities having lower molecular weights are present. The general protease activity elution profile is similar to the receptor-active protease profile for the 5-week-old rabbit but not the 13-week-old rabbit. The liver cytosol of the older animal has a high molecular weight protease active toward [14C]casein but not toward the estrogen receptor. The changes in the estrogen receptor forms and the receptor-modifying activity profiles of liver cytosol that occur during development in the rabbit suggest that receptor-modifying activity may initially be associated with the estrogen receptor to form a high molecular weight complex. 相似文献
17.
Secreted proteases from Photorhabdus luminescens: separation of the extracellular proteases from the insecticidal Tc toxin complexes 总被引:4,自引:0,他引:4
Bowen D Blackburn M Rocheleau T Grutzmacher C ffrench-Constant RH 《Insect biochemistry and molecular biology》2000,30(1):69-74
Photorhabdus luminescens secretes both high molecular weight insecticidal toxin complexes and also a range of extracellular proteases into culture broth. Previous studies by others have suggested that insecticidal activity of the broth is associated with these proteases. However, by gene cloning and targeted knock-out, we have previously shown that oral insecticidal activity is associated with high molecular weight 'toxin complexes' (Tc) encoded by toxin complex or tc genes. Here we further clarify this distinction by biochemically separating the protease fractions away from the oral insecticidal activity of the Tc proteins. We purified three distinct protease fractions from the broth: one consisting of a single species of 55 kDa and two of several putatively related species of approximately 40 kDa. All of these clearly separate from the oral insecticidal activity associated with the high molecular weight Tc proteins and also show no effect on insect weight gain following injection into the haemocoel. Here we examine the substrate preferences and inhibitor profiles of these protease fractions and discuss their relationship with those previously described from other P. luminescens strains and phase variants. 相似文献
18.
Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. 总被引:22,自引:3,他引:19
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C M Moehle R Tizard S K Lemmon J Smart E W Jones 《Molecular and cellular biology》1987,7(12):4390-4399
The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence. 相似文献
19.
Isolation of insulin degradation products from endosomes derived from intact rat liver 总被引:7,自引:0,他引:7
F G Hamel B I Posner J J Bergeron B H Frank W C Duckworth 《The Journal of biological chemistry》1988,263(14):6703-6708
Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease. 相似文献
20.
A high molecular weight protease inhibitor has been purified from the cell-free plasma of the horseshoe crab Limulus polyphemus using high speed centrifugation, polyethylene glycol precipitation, and gel filtration. The inhibitor is sensitive to mild acidification, methylamine treatment, and inhibits the proteolytic activity of a variety of endopeptidases. The molecule does not inhibit trypsin-mediated hydrolysis of low molecular weight substrates and protects the active site of trypsin from inactivation by soybean trypsin inhibitor. These properties are diagnostic of the alpha 2-macroglobulin (alpha 2M) class of protease inhibitors found in vertebrates. Like vertebrate alpha 2M the Limulus alpha 2M molecule is composed of subunits of molecular weight 180,000-185,000 as determined by polyacrylamide gel electrophoresis under reducing conditions. The apparent native molecular weight for the Limulus molecule as determined by both gel filtration and gel electrophoresis under nonreducing conditions is 500,000-550,000, compared to a native molecular weight of 700,000-750,000 for human alpha 2M, determined in parallel under identical conditions. These results suggest that alpha 2M appeared in evolution at least 550 million years ago before the divergence of the lineages that gave rise to present-day arthropods and mammals. 相似文献