Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we describe some modifications of the collagenase digestion technique for the preparation of pancreatic islets, that we have found helpful in preparing isolated islets of Langerhans from rabbits and hamsters. These modifications include disrupting the pancreatic acinar tissue by directly infiltrating it with Hanks' solution, periodically monitoring the progress of the collagenase digestion of the pancreatic tissue with the dissecting microscope, and purifying the islets by serial passage of the digested tissue through three Petri dishes containing Hanks' solution. This technique results in the successful isolation of the fragile and irregular rabbit islets as well as the isolation of the sturdier and more uniformly shaped hamster islets. The isolated respond to a glucose stimulus with an increase in radioimmunoassayable insulin secretion. We also describe a filtration technique for the preparation of isolated islets for histological examination. Isolated islets are retained on Millipore filters. The filters are then fixed, stained and mounted. Finally, we present realistic photographs of rabbit and hamster islets, as they appear under the dissecting microscope, during various stages of purification and isolation.
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PMID:Preparation of islets of Langerhans from rabbits and hamsters by the collagenase digestion technique. 17 31

Hydroxyproline-containing structural glycopeptide fractions were isolated from collagenase-digested neutral salt-insoluble collagen of five-day sponge-implant connective tissue of the rat. The glycopeptide fractions characterized migrate as a single, strongly anionic band on disc gel electrophoresis at pH 9.5, are eluted on gel filtration as a small molecular weight peak, approximately 2000, and are resolved into thirteen glycopeptide fractions by DEAE-cellulose chromatography. Amino acid analyses of some of these fractions indicate a similarity in composition, the principal ones being aspartic and glutamic acids, serine, glycine, alanine, valine, proline and hydroxyproline. Three neutral carbohydrates, glucose, mannose and xylose, in different relative proportions and hexosamine are also present in the fractions. Amino-terminal amino acid determinations indicate a microheterogeneity of the glycopeptides. The electrophoretic behaviour and non-diffusibility of the small molecular weight glycopeptides suggest an intimate association between acidic hydroxyproline-containing peptides and carbohydrate components of developing connective tissue.
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PMID:Hydroxyproline-containing structural glycopeptide fractions from subacute inflammation connective tissue. 17 78

During the digestion of pancreatic pieces with collagenase for prepartion of isolated islets the enzymes in incubation medium (collangenolytic and/or proteolytic) can alter the secretion behavior of A- and B-cells. Insulin release after such an enzymatic attack is characterized by an enhanced basal secretion and a diminished and delayed glucose response. Overdigestion results in a decreased glucagon secretion in response to arginine, a diminished insulin content, and a decreased thiol-protein-disulfide-oxidoreductase activity of the islets. Increased albumin concentrations did not prevent the collagenase effect.
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PMID:Investigations on isolated islets of Langerhans in vitro. XIII. Experiments concerning the preparation conditions with collagenase. 17 1

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.
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PMID:Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets. 18 Oct 79

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.
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PMID:The role of calcium ion in epinephrine activation of lipolysis. 18 5

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.
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PMID:A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate. 18 29

The effect of somatostatin on insulin secretion in response to a variety of stimuli was investigated in micro-dissected as well as collagenase isolated pancreatic rat islets. Somatostatin inhibited insulin secretion in both islet preparations in the presence of different initiators, but failed to affect the hormone output induced by theophylline, 1-methyl-3-isobutylxanthin (IBMX) or p-chloromercuriphenylsulfonic acid (CMBS). The inhibitory action was associated with decreased glucose metabolism by islets (measured as conversion of U-14C-glucose to 14CO2).
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PMID:Somatostatin-induced inhibition of insulin secretion by isolated pancreatic rat islets prepared by micro-dissection or collagenase digestion. 18 97

The uptake of 45Ca2+ by a lanthanum-non-displaceable pool in pancreatic islets was studied; Raising the extracellular D-glucose concentration from 3 to 20 mM stimulated the 45Ca2+ uptake in hand-dissected islets of ob/bo-mice as well as in collagenase-isolated islets of ob/ob or normal mice. The effect was dose-dependent in the range of 0-20 mM D-glucose and was seen throughout a wide range of extracellular calcium concentrations (16 mumol-2.56 mmol of Ca2+ added per litre of medium). The 45Ca2+ uptake was also enhanced by other known insulin secretagogues (D-mannose, L-leucine, tolbutamide) and was uninfluenced by compounds lacking insulin-releasing capacity (3-O-methyl-D-glucose, L-glucose, D-galactose, D-leucine). The stimulatory effect of D-glucose was blocked by inhibitors of glucose-induced insulin release (D-mannoheptulose, diazoxide, L-adrenaline). The results support the view that the lanthanum-nondisplaceable calcium pool is related to the insulin-releasing mechanism, although the exact nature of this relationship is still unclear.
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PMID:Effects of various modifiers of insulin release on the lanthanum-nondisplaceable 45Ca2+ uptake by isolated pancreatic islets. 19 76

The authors present morphological characteristics of primary monolayer cultures prepared from the pancreas of bovine fetuses. Combined treatment with trypsin and collalytine solutions (a preparation with collagenase activity) was used for dispersion of the tissue of the pancreas. Numerous epithelial cells corresponding by morphofunctional characteristics to beta-cells of the islets of Langerhans were contained in be cultures obtained; an aldehyde-fuchsin-positive granularity was revealed in the cytoplasm of these cells. Degranulation of these cells occurred under the effect of an increased glucose concentration in the nutrient medium.
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PMID:[Cell cultures obtained using collalytin from the pancreases of cattle fetuses]. 19 89

Glucose stimulation increased the cAMP content of collagenase-isolated rat pancreatic islets fourfold above baseline values. The elevation was transient, lasting about 5 min, and was dose-dependent. Insulin release continued at a constant rate throughout the incubation. Glucagon, in the absence of glucose, increased cAMP for about 1 min but only slightly, and had no effect on insulin release. In the presence of glucose, however, glucagon enhanced islet cAMP content 15-fold and increased the release of insulin. Glucagon was most effective at high glucose concentrations (16.6 and 25 mM). This indicates that glucagon is critically dependent on the presence of glucose in order to increase the islet cAMP content and to stimulate insulin release. The inability of glucagon to generate sufficient cAMP in the absence of glucose might be one of the reasons why the hormone is a potentiator rather than an initiator of insulin release.
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PMID:Permissive effect of glucose on the glucagon-induced accumulation of cAMP in isolated rat pancreatic islets. 19 21


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