The kinetic parameters of three activator species of Glu1-plasminogen (Glu1-Plg) were compared in their reaction at pH 7.4 and 37 degrees C, in the presence and absence of CNBr-digested fibrinogen (CNBr-Fg). The urokinase- (u-PA-) derived covalent hybrid activator PlnA-u-PAB had an apparent Michaelis constant (Kplg) of 7.44 microM, a catalytic rate constant (kplg) of 51.1 min-1, and a second-order rate constant (kplg/Kplg) of 6.87 microM-1 min-1. The tissue plasminogen activator (t-PA) derived covalent hybrid activator PlnA-t-PAB was characterized by a Kplg of 3.33 microM, a kplg of 1.03 min-1, and a kplg/Kplg of 0.309 microM-1 min-1. The kplg/Kplg values for the parent u-PA and t-PA activators were 6- and 16-fold higher than the respective hybrids, mainly due to an approximately 10-fold increase in the apparent Kplg for the hybrids. In the presence of CNBr-Fg, the increase of the kplg/Kplg values for u-PA and its hybrid was 1.1-fold, but for t-PA and its hybrid, the increases were 7- and 12-fold, respectively. In both the absence and presence of CNBr-Fg, activator t-PAB had an apparent Kplg of 19.1 and 27.6 microM and a kplg of 2.9 and 5.0 min-1, respectively. The increase in the kplg/Kplg value with CNBr-Fg was 1.2-fold. The streptokinase- (SK-) derived activators Glu1-plasmin.SK (Glu1-Pln.SK), Val442-Pln.SK, and Val561-Pln.SK had apparent Kplg values of 0.458, 0.268, and 0.121 microM and kplg values of 20.0, 126.0, and 63.3 min-1, respectively. In the presence of CNBr-Fg, the first two activators showed an approximately 1.4-fold increase and the last showed a 1.4-fold decrease in their kplg/Kplg values. The catalytic efficiency (kplg/Kplg) of the various activator species fell in the decreasing order SK greater than u-PA greater than t-PA, in either the presence or absence of CNBr-Fg. CNBr-Fg enhanced significantly the activities of only two activators, t-PA and PlnA-t-PAB. 相似文献
4-Methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine oligosaccharides were used as substrates to detect expression of a Streptomyces chitinase in Escherichia coli. Low levels of enzyme were detected when S. plicatus DNA was cloned into a bacteriophage lambda vector (EMBL-4). Subcloning into E. coli plasmids also gave low but detectable levels of enzyme expression. High level expression was achieved by resection of the cloned S. plicatus DNA with Bal31 followed by in-frame fusion to the amino-terminal peptide sequence of beta-galactosidase found in the pUC vectors. The Streptomyces chitinase was secreted into the periplasmic space of E. coli, and its signal sequence was removed. We characterized the activity of the cloned enzyme and compared it to three other purified Streptomyces plicatus chitinases with respect to hydrolysis of the 4-MU oligosaccharides. We found that two of the enzymes form 4-methylumbelliferone much more rapidly from the 4-MU disaccharide than from the trisaccharide. These same enzymes convert the 4-MU trisaccharide primarily to diacetylchitobiose and the 4-MU monosaccharide, a nonfluorescent product. The latter compound is not hydrolyzed appreciably by any of the enzymes. On the basis of these results, we suggest a new definition of "exo" and "endo" chitinase that differs from that found in the literature. We propose that exochitinase activity be defined as processive action starting at the nonreducing ends of chitin chains with release of successive diacetylchitobiose units, and that endochitinase activity be defined as random cleavage at internal points in chitin chains. 相似文献
Native x-ray diffraction data from single crystals of inactive aconitase from pig heart (Mr 80,000) have been collected on oscillation films to 2.7 A. Analysis shows that significant measurements of the anomalous scattering signal from the Fe-S cluster in the enzyme are available in the film data. The 5.0-A resolution anomalous difference Patterson function contains vectors for one Fe-S cluster (one aconitase molecule) per asymmetric unit in space group P2(1)2(1)2 with a = 173.6, b = 72.0, and c = 72.7 A. At 2.7-A resolution, the vector map is best interpreted by three Fe sites separated from each other by less than 3 A. The single-crystal diffraction data thus confirm the presence of a 3Fe center in the inactive form of aconitase. Furthermore, the data provide crystallographic evidence that 3Fe clusters exhibit structural heterogeneity. The Fe-Fe vectors cannot be interpreted in terms of 4-A distances as observed for the [3Fe-3S] cluster in Azotobacter ferrodoxin (Ghosh, D., O'Donnell, S., Furey, W., Robbins, A. H., and Stout, C. D. (1982) J. Mol. Biol. 158, 73-109). The results are therefore in agreement with a [3Fe-4S] cluster having 2.7-A Fe-Fe distances (Beinert, H., Emptage, M. H., Dreyer, J.-L., Scott, R. A., Hahn, J. E., Hodgson, K. O., and Thomson, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 393-396). However, the data do not unambiguously discriminate between this model and other 3Fe clusters having short Fe-Fe distances. 相似文献
The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants. 相似文献
The v-sis transforming gene encodes the woolly monkey homologue of human platelet-derived growth factor (PDGF) polypeptide 2. After its synthesis on membrane bound polyribosomes, the glycosylated precursor dimerizes in the endoplasmic reticulum and travels through the Golgi apparatus. At the cell periphery, the precursor is processed to yield a dimer structurally analogous to biologically active PDGF. Small amounts of two incompletely processed forms are detectable in tissue culture fluids of simian sarcoma virus (SSV) transformants. However, the vast majority remains cell associated. Thus, this growth factor-related transforming gene product is not a classical secretory protein. These findings define possible cellular locations where the transforming activity of the sis-PDGF-2 protein may be exerted. 相似文献
Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length. 相似文献
Murine antibody responses to heterologous insulins are under H-2-linked immune response (Ir) gene control. We have found that the immune response to insulin in adjuvant can be inhibited by prior i.v. injection of soluble insulin. The effect of i.v. injection of insulin is antigen-specific and dose-dependent and requires the same doses of insulin that are immunogenic if administered with adjuvant. In addition, the inhibitory effect of soluble insulin is dependent upon the route of injection; if soluble insulin is injected i.p., the subsequent response to insulin in adjuvant is augmented rather than inhibited. Unresponsiveness requires at least 4 days after i.v. injection to develop and once induced, it is maintained for 4 wk or more. Unresponsiveness is caused by T cell, but not B cell, tolerance, and we have been unable to demonstrate any role for suppressor T cells in this unresponsiveness. More importantly, analysis of the ability of numerous insulin variants to induce unresponsiveness in several H-2k and H-2b strains of mice has demonstrated that only the variants that were immunogenic in a given strain when administered with adjuvant were able to cause tolerance. This report is, to our knowledge, the first describing that induction of helper T cell tolerance, like the induction of immunity, is controlled by H-2-linked Ir genes. 相似文献
Summary A transitory cell division block, or excision effect, occurs in the meristem of roots after excision and transfer to culture medium. This block can be induced, in intact seedling roots, by exogenous treatment with ethylene gas. With continuous treatment, the block is longer and the recovery less than after a 4 hour pulse. In excised roots the excision effect can be eliminated by treatment with an inhibitor of ethylene synthesis (aminoethoxyvinylglycine) or action (silver thiosulfate). These experiments provide evidence to support the hypothesis that ethylene from the wounded end of an excised root is involved in a process resulting in a transitory block in cell cycle progression in the meristem. The implications of this hypothesis are discussed. 相似文献
A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]5[NAcG1cNH2]2-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]3[Man]9[NAcG1cNH2]2P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins. 相似文献
We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.
All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.
The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.