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1.
We have isolated mutant strains of the cellular slime mold, Dictyostelium discoideum, which specifically lack α-mannosidase-1. This enzyme accumulates during the developmental phase of the life cycle. A minor isozyme, α-mannosidase-2, was recognized which accumulates only during culmination and makes up about 10 p. cent of the total activity in fruiting bodies. The minor isozyme has a pH response distinctly different from the major isozyme. Loss of the major isozyme, α-mannosidase-1, does not disrupt the normal sequence of biochemical differentiations. 相似文献
2.
Ellen Van Obberghen-Schilling Jacques Pouyssgur 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,847(3):335-343
The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of Mr ≈ 150 000 revealed as a 125I-α-thrombin cross-linked complex of Mr ≈ 180 000. Independent of chemical cross-linking, 125I-α-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response. 相似文献
3.
5-enolpyruvylshikimate 3-phosphate synthase, the target enzyme of the herbicide glyphosate, is synthesized as a precursor in a higher plant
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Cell cultures of Corydalis sempervirens adapted to growth in the presence of 5 millimolar glyphosate overproduce the herbicide's target enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, 30- to 40-fold. In vitro translation of total RNA and poly(A)-RNA coupled with immunoprecipitation showed that the protein is synthesized as a precursor of relative molecular weight (Mr) 53900 ± 900 as compared to Mr 45500 ± 1000 of the mature enzyme. Translatable activity of mRNA for EPSP-synthase in glyphosate-adapted cultures is tenfold higher than in nonadapted cultures. 相似文献
4.
Marie-France Champliaud Ismo Virtanen Car-Fredrik Tiger Matti Korhonen Robert Burgeson Donald Gullberg 《Experimental cell research》2000,259(2):326
Laminins assemble into trimers composed of α, β, and γ chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin α1 and α5 chains. Comparative pulse–chase experiments and deglycosylation studies in JAR cells established that the Mr 360,000 laminin α1 chain is glycosylated into a mature Mr 400,000 band while the Mr 370,000 laminin α5 chain is glycosylated into a Mr 390,000 form that upon secretion is further processed into a Mr 380,000 form. Hence, despite the shorter peptide length of α1 chain in comparison with the α5 chain, secreted α1 assumes a larger size in SDS–PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin α1 and laminin α5 chains in laminin-1 and laminin-10. In placenta laminin α1 chain (Mr 400,000) and laminin α5 chain (Mr 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin α1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin β2 chains. Surprisingly, a fraction of the laminin α1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin β1–β3 chains, possibly pointing to an unexpected complexity in the chain composition of α1-containing laminin isoforms. 相似文献
5.
Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum 总被引:3,自引:0,他引:3
The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development. 相似文献
6.
Hiroyuki Inoue Akira Nishito Shin-ich Eriguchi Takashi Tamura Kenji Inagaki Hidehiko Tanaka 《Journal of Molecular Catalysis .B, Enzymatic》2003,23(2-6):265
α-Ketobutyrate decarboxylase encoded in the
-methionine catabolism operon of Pseudomonas putida is homologous with the E1 component of pyruvate dehydrogenase complex from gram-negative bacteria. The enzyme was purified to homogeneity from the cell extract of an Escherichia coli transformant. The purified enzyme was homodimeric with a subunit of Mr 93,000 on SDS-PAGE. The enzyme activity was activated by the addition of both thiamine pyrophosphate (TPP) and a divalent cation, such as Mg2+, Mn2+, and Co2+. The enzyme showed high activity for α-ketobutyrate and α-keto-n-valerate rather than pyruvate, but the α-keto acids with increasing length of the side chain as well as branching, such as α-keto-n-caproate and α-keto-3-methylvalerate, were not used by the enzyme. The Km values for α-ketobutyrate and pyruvate were 0.016 and 0.147 mM, respectively, and the kcat/Km value (10.69 s−1 mM−1) for α-ketobutyrate was 29-fold greater than that for pyruvate. Thus, α-ketobutyrate decarboxylase is distinguished from the pyruvate dehydrogenase E1 component with respect to the substrate specificity, although their structural and enzymological properties were similar. These results suggest that the unique substrate specificity of α-ketobutyrate decarboxylase is due to a slight difference in the highly conserved active sites of both enzymes. 相似文献
7.
We have purified a unique enzyme, α-amino--caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with Mr 50 000 and a sedimentation coefficient (s20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is 9.0. D- and L-α-amino--caprolactams are the only substrates. The Km values for the D- and L-isomers are, 8 and 6 mM, respectively. 相似文献
8.
γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195 000±10 000 in its dimeric form with an Mr of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme. 相似文献
9.
Richard A. Hiipakka Marjorie Wang Tim Bloss Kiyoshi Ito Shutsung Liao 《The Journal of steroid biochemistry and molecular biology》1993,45(6)
A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with Mr 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a Mr 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a Mr 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei. 相似文献
10.
Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were α-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr > 25 000). Over a 2 h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25 000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional ‘holes’ of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and non-confluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional ‘holes’ of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death. 相似文献
11.
Anne-Ccile Cotillon Jaleh Doostzadeh Robert Morfin 《The Journal of steroid biochemistry and molecular biology》1997,62(5-6)
7α-Hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7α-position, with minor hydroxylation occurring at the 7β-position. Constitutive 7α-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7α-hydroxylation via an increase in protein synthesis. DHEA 7α-hydroxylase was found to be mainly microsomal, and the best production yields of 7α-hydroxy-DHEA (28.5 ± 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (KM=1.18 ± 0.035 μM and Vmax=909 ± 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7α-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7α-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7α-hydroxylase; (iii) this enzyme complex contains a cytochrome P450. 相似文献
12.
Adele R. Thomas Ryno J. Naud Willem Oelofsen Takako Naganuma Koji Muramoto 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,129(4)
This study reports the isolation and partial characterisation of the ostrich serpin, α2AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich α2AP was purified using
lysine–Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI–Sepharose chromatographies. It revealed a Mr of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine α2AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human α2AP, DFP and EACA. Ostrich plasminogen was highly purified after
lysine–Sepharose chromatography and showed a Mr of 92 K, a total of 775 amino acids and its N-terminal sequence showed 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a Mr of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40°C, respectively. 相似文献
13.
14.
Nicolas Boisset Franois Pochon Serge Chwetzoff Martine Barray Etienne Delain Jean Lamy 《Journal of structural biology》1992,108(3)
In order to covalently bind the hydrolyzed thiol ester groups of the human α2-macroglobulin (α2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (Mr = 13 000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed α2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of α2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in α2M. They also suggest that the four thiol esters are located near the center of the α2M. molecule. 相似文献
15.
16.
Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced 125I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of Mr =116 and 80 kDa and a minor band of Mr =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes. 相似文献
17.
Guy Marlas 《Biochimie》1985,67(12)
It was concluded in a previous paper [13] that the high Mr platelet-activating glycoprotein isolated earlier from the venom of Crotalus durissus cascavella [11–12] has an hexameric structure of the α3β3 type involving two distinct subunits. Data reported here demonstrate that these two subunits are separable from each other by ion exchange chromatography under denaturating conditions, have similar Mrs (α = 12,540 et β = 13,770) and exist in a one to one ratio within the native molecule. Carbohydrate analysis indicated that they are both similarly glycosylated to a small extent. They have slightly different amino-acid compositions, a common N-terminal sequence up to the fifth residue and similar extinction coefficients at 280 nm. The native molecule has a calculated Mr of 78,930. Additional data demonstrated that convulxin from the venom of Crotalus durissus terrificus [3] is the same platelet-activating agent as the presently described platelet-activating glycoprotein (PAG) from the venom of Crotalus durissus cascavella [11–13]. 相似文献
18.
Susan A. Moore 《Experimental cell research》1987,171(2)
A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t1/2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, KI, represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10−10M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K50 (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10−8M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant KD and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10−8M and 104, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 nM. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed. 相似文献
19.
Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low Mr (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium Mr (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low Mr agarans or agaran-dl-hybrids. 相似文献
20.
The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH2-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC)in vitro.There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330–410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1α, IL-1β, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280–295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P< 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1α and IL-1β increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1α, IL-1β, TNF-α, and IL-8. 相似文献