共查询到20条相似文献,搜索用时 15 毫秒
1.
In agreement with previous glucose binding data, kinetic studies of the yeast hexokinases at high protein concentration show that the dimeric forms P-I and P-II require much higher glucose concentrations for half-maximal rates than do the monomeric forms S-I and S-II, but each P form reaches the same V value as the corresponding S form. Raising the temperature from 5 °C to 24 °C causes an apparent dissociation of the P forms, most pronounced in the case of P-I, as evidenced by a lowering of glucose KM values toward those of the S forms. Similarly, kinetic measurements at low protein concentrations, where the P forms are presumably fully dissociated, give identical glucose KM values for the P and S forms. 相似文献
2.
The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity. 相似文献
3.
Chemistry and subunit structure of yeast hexokinase isoenzymes 总被引:4,自引:0,他引:4
Evidence from ultracentrifugation, sodium dodecyl sulfate electrophoresis, peptide mapping, and carboxypeptidase A digestion allows the conclusion that the two native hexokinases, P-I and P-II, consist of polypeptide chains having molecular weights slightly higher than 50,000. It was demonstrated that some preparations are contaminated with a protease, and that this impurity caused erroneous results in sodium dodecyl sulfate electrophoresis and carboxypeptidase A digestion.Amino acid analyses indicated that both P-I and P-II contain four cysteine, four tryptophan, and eleven methionine residues per mole. In contrast, P-I contains eight, and P-II five, histidine residues per mole. Many of the differences in amino acid composition are small, but reproducible.Peptide mapping indicated that many segments of P-I and P-II have identical sequences. There were about 27 common tryptic peptides, and about 16–19 unique to each form. In addition, both isozymes were found to have the same amino terminus, valine, and the same carboxy terminus, alanine; some evidence for a difference in the penultimate residue at the carboxy terminus was indicated. 相似文献
4.
Structural relationships of the three stimulatory factors of RNA polymerase II from Ehrlich ascites tumor cells 总被引:6,自引:0,他引:6
M Horikoshi K Sekimizu S Hirashima Y Mitsuhashi S Natori 《The Journal of biological chemistry》1985,260(9):5739-5744
The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment. 相似文献
5.
F C Womack M K Welch J Nielsen S P Colowick 《Archives of biochemistry and biophysics》1973,158(2):451-457
An improved procedure for purification of the hexokinases P-I and P-II from baker's yeast is described. Yields, reproducibility, and purity are improved over those found by the methods used previously in this laboratory. The growth of large crystals of form P-I is described.Antisera prepared against the two purified hexokinases show only slight cross reaction by microcomplement fixation. The anti-sera have been used to demonstrate the presence of both P-I and P-II in crude extracts of various yeasts, including two haploid strains, and their absence in a yeast which contains glucokinase but no hexokinases. 相似文献
6.
7.
T Nakanishi A Nakano K Nomura K Sekimizu S Natori 《The Journal of biological chemistry》1992,267(19):13200-13204
Saccharomyces cerevisiae S-II was purified to near homogeneity as a protein stimulating RNA polymerase II. Four of seven lysyl endopeptidase-digested fragments of S-II were located in the PPR2 sequence reported previously. Analysis of a genomic clone of S-II revealed that S-II and PPR2 are the same protein consisting of 309 amino acid residues, and frame shifts were found in the sequence of PPR2 gene reported previously. Yeast S-II and mouse S-II showed high similarity in their amino acid sequences, especially in their amino-terminal and carboxyl-terminal regions. A gene disruption experiment showed that an S-II null mutant was not lethal under usual growth conditions, indicating that S-II is not essential for the growth of yeast. 相似文献
8.
P Ruth W Landgraf A Keilbach B May C Egleme F Hofmann 《European journal of biochemistry》1991,202(3):1339-1344
cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level. 相似文献
9.
Hitoshi Saito Hiromi Yamada Yoshiomi Kato 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,119(4):625-630
A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects. 相似文献
10.
Saurabh Pandit Kirti Shitiz Hemant Sood Rajinder Singh Chauhan 《Journal of plant biochemistry and biotechnology.》2013,22(3):335-342
Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the North-Western Himalayas. Extensive harvesting for pharmaceutical purposes, lack of organized cultivation and unorganized methods of uprooting the plants because of unawareness has brought an endangered status to this important herb in nature. The medicinal property of this plant is attributed to monoterpenoid picrosides. The influence of developmental status of different growth stages on picrosides content is poorly understood in Picrorhiza kurroa. Picroside-I (P-I) content increased from 0.05 % to 0.76 % in different growth stages of shoots. Significant increase in the contents of P-I (0.15–0.50 %) and Picroside-II (P-II) (0.1–0.45 %) was observed in rhizomes of different developmental stages. Highest amounts of P-I (8.7 %) and P-II (5.3 %) was detected in uppermost part of mature dried rhizomes compared to bottom part with 2.9 % and 2.2 % of P-I and P-II, respectively. P. kurroa grown at high altitude (Sairopa, 4,500 amsl) showed 1.75-folds increase in P-I in leaves whereas exponential increase in the P-I content was detected (0.05–1.7 %) in the leaves of different developmental stages (L1-L5) of P. kurroa grown at lower altitude (Jagatsukh, 1,900 m). Variable amounts of P-I and P-II in different growth and developmental stages of P. kurroa imply importance of selection of plant material (rhizomes and roots). The study undertaken explored the status of metabolites accumulation and biosynthesis in the field grown plants of P. kurroa where not only environmental parameters but different morphogenetic stages of its developmental cycles, different age groups and different parts of plantlets were extensively analysed and estimated for medicinally important picrosides. 相似文献
11.
Gurrinder Singh Bedi 《Preparative biochemistry & biotechnology》2013,43(2):85-104
Abstract The present investigation describes the comparative properties, particularly the substrate specificity of three kallikrein-like serine proteinases (I, II and III) purified from rat submandibular gland extract (Bedi, G.S., Prep. Biochem. 22, 67–81. 1992). The physico-chemical and immunological properties of three proteinases were compared by Western blot analysis, immunodiffusion, immuno-electrophoresis, amino terminal sequence analysis, molecular weight determination and isoelectric focusing. Detailed substrate specificity of these proteinases was determined using chromogenic substrates, synthetic peptides and native proteins. The chromogenic substrate tosyl-gly-pro-arg-pNA was hydrolyzed preferentially by Proteinase I. The replacement of pro at the P2 position with bulky hydrophobic residues phe and leu completely abolished the hydrolysis by Proteinase I. The hydrolysis of the chromogenic substrates by Proteinase II was also affected by the amino acid residue present at the P2 position in the order of pro>gly>val>leu>phe. Neither Proteinase I nor Proteinase II hydrolyzed substrates in which arg was replaced with lys at the P1 position. Proteinase III was reactive against all the chromogenic substrates with arg or lys at the P1 position. Synthetic polypeptides T-kinin-leu and insulin B chain were resistant to cleavage by both Proteinase I and II and were cleaved specifically at arg-X peptide bond by Proteinase III. Tonin-like activity of Proteinase II was confirmed by cleavage of the angiotensin 1–14 at phe-his linkage to generate two fragments DRVYIHPF and HLLVYS respectively. All three proteinases cleaved human high molecular weight kininogen but only Proteinase III could cleave T-kininogen. Proteinase III was also reactive towards human and bovine fibronectin, fibrinogen and gelatin. Several other salivary and serum proteins were resistant to cleavage by these proteinases. Although the three enzymes are immunologically related, they differ with respect to size, isoelectric point, amino terminal sequence and inhibition profile. 相似文献
12.
Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall. 相似文献
13.
Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis. 相似文献
14.
15.
Partial digestion of the native beta subunit of F1-ATPase from the thermophilic Bacillus strain PS3 by three different proteases produced a limited number of peptide fragments. In most cases, the peptides remained associated, and the gross structure of the beta subunit was not destroyed. Furthermore, most peptides were able to reassociate into the form of the beta subunit after denaturating urea treatment. Therefore, the cleaved sites are most likely located in water-exposed loop regions in the tertiary structure of the protein. Almost all peptides were analyzed, and 17 cleaved sites were determined. From the analysis of the distribution of cleaved sites and deletions or insertions in the multiple amino acid sequence alignment of proteins homologous to the beta subunit, locations of five loops and four candidate loops in the beta subunit are suggested. There are two large loops in the central region of the beta subunit sequence, and dicyclohexylcarbodiimide-reactive Glu190 is located in one of them. Tyr341, involved in putative catalytic ATP binding, is also found in one of the loops. Then, taking cleaved sites as a reference, two kinds of expression plasmids, each of which carried genes of two complementary peptide fragments, 1-193 and 198-473 or 1-284 and 285-473, were constructed and expressed in Escherichia coli. For each plasmid, two peptides were coexpressed, associated into a stable beta subunit form in E. coli cells, and purified without dissociation. When these beta subunits were denatured by urea and applied to polyacrylamide gel without denaturant, a protein band with the same mobility as that of the beta subunit appeared, indicating that reassociation of peptide fragments into the form of the beta subunit occurred upon removal of urea. These beta subunits retained the ability to reconstitute the alpha 3 beta 3 gamma complexes even though the efficiency of reconstitution and the recovered ATPase activities were decreased. These complexes were stable at high or low temperature, and ATPase activities were sensitive to inhibition by N3-. 相似文献
16.
Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed. 相似文献
17.
Zhang X Ma C Tang J Tang W Tu J Shen J Fu T 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2008,117(2):171-179
Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding. 相似文献
18.
19.
Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S-1.M-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification. 相似文献
20.
Three basic proline-rich peptides were newly isolated from human parotid saliva, and designated as P-G, P-H, and P-I. The amino acid sequence of P-H was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Glu-Gly-Asn-Asn- Pro-Gln-Gly-Pro-Pro-Pro-Pro-Ala-Gly-Gly-Asn-Pro-Gln-Gln-Pro-Gln-Ala-Pro-Pro- Ala-Gly-Gln-Pro-Gln-Gly-Pro-Pro-Arg-Pro-Pro-Gln-Gly-Gly-Arg-Pro-Ser-Arg-Pro- Pro-Gln by conventional methods. The amino terminal ten residues of P-H were the same as those of proline-rich peptides P-D, P-E, and P-F reported previously. Comparison of the amino acid sequences between P-H and P-D revealed that there are two deletion parts and several amino acid substitutions in the sequence of P-H. Homology between P-H and P-D was as high as 70%. 相似文献