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41.
42.
43.
Shun-Ichiro Kawabata Takashi Morita Toshiyuki Miyata Shigenori Kaida Sadaaki Iwanaga Hideo Igarashi 《Journal of Protein Chemistry》1987,6(1):17-32
The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (Kd=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (Kd=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985. 相似文献
44.
E P Beem J J W Lisman G J Van Steijn C J Van Der Wal L A W Trippelvitz B Overdijk H Van Halbeek J H G M Mutsaers J F G Vliegenthart 《Glycoconjugate journal》1987,4(1):33-42
Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kDa and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz1H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man5–8GlcNAc2), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified. 相似文献
45.
The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions. 相似文献
46.
Summary Transforming growth factor beta (TGF-) activates ATP citrate lyase in freshly isolated rat liver hepatocytes in a time dependent manner. Maximal stimulation of the enzyme occurred with less than thirty minutes of incubation of the cells with TGF-. The half maximal effect on the enzyme determined in hepatocytes incubated with TGF- for 10 min at 37°C was elicited by TGF- concentrations in the 10–11 – 10–12 M range. The potential role of TGF- stimulation of ATP citrate lyase activity in new membrane synthesis is discussed. 相似文献
47.
Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations
AChE
acetylcholinesterase
-
BChE
butyrylcholinesterase
-
iso-OMPA
specific inhibitor of BChE
-
BW 284C51
specific inhibitor of AChE
-
IPL
inner plexiform layer
-
OPL
outer plexiform layer 相似文献
48.
Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified. 相似文献
49.
Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin 总被引:24,自引:0,他引:24
The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM
Black lipid membranes
- CCCP
carbonyl cyanide m-chlorophenylhydrazone
- DOPC
dioleoyl phosphatidylcholine
- PS
phosphatidylserine
- TPP+
tetraphenylphosphonium cation 相似文献
50.
J. Ruiz-Herrera J. P. Martinez M. Casanova M. L. Gil R. Sentandreu 《Archives of microbiology》1987,149(2):156-162
Cells from the slime variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER
Endoplasmic reticulum
- UDP-GlcNAc
uridine-5-diphosphate N-acetyl glucosamine
- GlcNAc
N-acetyl glucosamine
- SDS
sodium dodecyl sulfate
- PMSF
phenyl methyl sulfonyl fluoride
- EDTA
ethylene diamino tetraacetic acid
Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico 相似文献