Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.
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PMID:Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds. 226 95

We examined the qualitative patterns of protein synthesis in fully grown prophase-blocked oocytes of Xenopus laevis and after meiosis reinitiation accompanying maturation of the oocytes. Newly synthesized proteins labelled with [35S]methionine were run on isoelectric focusing gels and further separated in the second dimension on SDS-polyacrylamide slab gels. Three types of maturation inducer were compared: progesterone, considered as the natural inducer of Xenopus oocyte maturation, hCG (human chorionic gonadotropin) and insulin. Three polypeptides with apparent molecular masses of 37 kDa (pI 4.7-4.8), 78 kDa (pI 4.7) and 138 kDa (pI 4.6-4.7) were found to be always synthesized in all three types of stimulation, while the synthesis of a fourth one (molecular mass 116 kDa, pI 4.7) was arrested during oocyte maturation. Moreover, when the follicular cells surrounding the oocytes were part of the stimulating pathway, which is the case during hCG-induced maturation, an additional polypeptide was synthesized by the oocytes (molecular mass 106 kDa, pI 6.0-6.2). This polypeptide was not synthesized during progesterone- or insulin-induced oocyte maturation, two types of stimulation which do not require the presence of the follicular cells. The biological significance of the hCG-induced polypeptide, not necessary for oocyte maturation, is discussed. On the other hand, the four other modifications in protein synthesis taking place during all three types of maturation-inducing stimulation appear to be necessary for oocyte maturation, since oocytes which failed to mature in response to stimulation always missed one or several of these four polypeptides.
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PMID:Patterns of protein synthesis during Xenopus oocyte maturation differ according to the type of stimulation. 227 96

An insulin receptor was found on the surface of cloned mouse glomerular endothelial cells in vitro. Total specific binding was 2.5 +/- 0.3%/10(6) cells at 90 min and 22 degrees C. Analysis according to Scatchard resulted in a curvilinear plot, with a kd for the high and low affinity sites estimated at 1.41 x 10(-10) and 8.2 x 10(-8) respectively. Insulin binding decreased following 12 hour exposure to 50 ng/ml of insulin suggesting that down regulation of the receptor had occurred, an effect which was reversible. Covalent crosslinking of the receptor to 125I insulin revealed one band at Mr 125,000 by SDS-PAGE which disappeared following preincubation with excess unlabeled insulin. Insulin was also able to stimulate phosphorylation of the beta subunit. The characteristics of this insulin receptor appear very similar to that of endothelial cell types from other microvascular beds.
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PMID:Mouse glomerular endothelial cells have an insulin receptor. 227 1

1. Lugworm protease C further purified by benzamidine-affinity chromatography, exhibited peptidase specificity for arginyl and lysyl bonds. 2. Protease C consisted of a single polypeptide with a mol. wt of ca 23,000 as determined by SDS polyacrylamide gel electrophoresis, exhibited a u.v. absorption maximum at 280 with an (mg/ml) extinction coefficient of 0.93 and fluorescence spectra typical of a protein containing tryptophan, and had an amino acid composition similar to trypsins. 3. The Kms of the cleavages of the arginyl bond of oxidized insulin B chain and of the lysyl bond of the gly23-ala30 fragment were determined to be 0.72 and 0.96 mM; the corresponding kcats were 38 sec-1 and 1.5 sec-1. The Km and kcat for TAME were 0.042 mM, and 110 sec-1. 4. Lugworm protease C was confirmed to be a trypsin.
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PMID:Characterization and peptidase specificity of lugworm (Arenicola cristata) protease C. 234 31

A partially purified insulin receptor preparation from rat liver was incubated at 37 degrees C with and without the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2/5.3.4.1). Insulin-binding activity was then assessed by crosslinking receptor-125I-insulin complexes and subjecting them to electrophoresis on SDS-polyacrylamide gels in the absence and presence of reductant followed by autoradiography. Prior incubation of the receptor at 37 degrees C in the absence of the enzyme markedly decreased the subsequent binding of 125I-insulin to the holoreceptor (Mr 350 000) and to its subunits (Mr 180 000 and 130 000), while addition of the enzyme to the preincubation medium served to substantially prevent this decrease. The loss in binding at 37 degrees C was not restored by subsequent addition of the enzyme, nor was the loss prevented by any of the several known inhibitors of proteolysis. The apparent stabilization of receptor by transhydrogenase, as evidenced by the increase in binding above control levels, was proportional to both the enzyme concentration and the duration of incubation. These effects seem to be specific for transhydrogenase, since several other disulfide-containing proteins were found to be ineffective. These data suggest that the stabilization of the subunit structure of the insulin receptor at physiological temperatures may take place via a disulfide interchange reaction catalyzed by glutathione-insulin transhydrogenase.
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PMID:Stabilization of insulin receptor subunit structure by glutathione-insulin transhydrogenase. 242 78

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.
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PMID:Production and characterization of monoclonal antibodies to insulin secretory granule membranes. 244 18

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-200 gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed-phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.
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PMID:Myelin basic protein present in the acid extracts of rat hypothalami releases insulin and glucagon from isolated rat pancreatic islets. 246 73

Both adrenalectomy and chemically induced diabetes mellitus cause a marked decrease of pancreatic amylase activity in rats, but it is unknown whether these effects are the result of a direct or indirect mechanism. The synthesis of various pancreatic enzymes has been studied in isolated pancreatic acini from sham-operated, castrated, and adrenalectomized animals as well as in animals that have been both adrenalectomized and castrated. Protein synthesis was measured by pulse labeling of acini with [35S]methionine followed by either trichloroacetic acid precipitation of total protein and counting or by SDS-PAGE and autoradiography, and additionally by in vitro translation of extracted pancreatic RNA using rabbit reticulocytes. Adrenalectomy resulted in a 70% reduction of amylase activity per milligram of acinar protein as a result of a decrease in amylase synthesis. This reduction in amylase synthesis is a consequence of a decrease in the amount of mRNA coding for amylase. After adrenalectomy, plasma concentrations of the following were reduced compared to controls: corticosterone to 0.45%, insulin to 11%, and glucose to approximately 66%. Addition of glucose to the drinking water caused an increase in insulin and plasma glucose, but this was not followed by an increase in amylase activity. We postulate that corticosterone directly regulates amylase synthesis in the rat pancreas.
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PMID:Role of glucocorticosteroids in the regulation of pancreatic amylase synthesis. 247 63

Insulin-like growth factors (IGFs) are bound to specific binding proteins in extracellular fluids in vivo and when released by cells in vitro. One class of binding protein (IGF-BP), a peptide of 26 kDa purified from amniotic fluid, has been shown to modulate IGF bioactivity on isolated human fibroblasts. We have determined the factors that control release of IGF-BP from monolayers of human fetal fibroblasts using a radioimmunoassay, and have compared this with the effects of these factors on the release of IGF-I and -II. Separation of cell-conditioned cultured medium on SDS-PAGE, and subsequent immunoblotting with antibody against IGF-BP showed that fibroblasts released a single species of immunoreactive protein of an estimated molecular weight of 30 kDa. This was not the predominant binding protein released by cells since major bands of approximately 42 kDa and 39 kDa were visualized following separation by SDS-PAGE and ligand blotting with 125I-labelled IGF-I. The 30 kDa IGF-BP was released in parallel with radioimmunoassayable IGF-I and -II over 48 h. However, a significant inverse correlation was found between the release of IGF-BP, IGF-I, IGF-II and cell density. The exposure of fibroblasts to 1.3 nmol/l or greater of IGF-I or -II caused a significant release of IGF-BP. Maximum release was seen in sparse cultures with little or no release from confluent cultures. IGF-I and -II were approximately equipotent with a fourfold increase in IGF-BP release at 19.7 nmol/l. Insulin caused a release of IGF-BP and IGF-I and -II from fibroblasts at supraphysiological concentrations (16.7 nmol/l) which again was maximal on sparse cell cultures. Increases in IGF-BP, IGF-I and -II release were also found in the presence of human placental lactogen (23.3 nmol/l), but human GH, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor were without effect. The results show that human fetal fibroblasts released an IGF-BP immunologically similar to that seen in amniotic fluid together with IGF-I and -II, that IGF-BP release was enhanced by exogenous addition of IGF peptides, and that the release of all three peptides was a property of sparsely plated, rapidly growing cells. These findings strengthen the hypothesis that the cellular expression of IGF-binding proteins may represent an important level of control in IGF physiology.
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PMID:Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasts: dependency on cell density and IGF peptides. 247 66

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with gamma 32P-ATP after treatment with EGF for 30 min to 3 h at 37 degrees C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis. 247 79


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