Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

1. Tubule fragments were isolated by collagenase treatment of guinea pig kidney cortex. 2. 3':5'-Cyclic AMP increased gluconeogenesis from lactate, pyruvate, malate and fructose. 3. Noradrenaline decreased gluconeogenesis from lactate, pyruvate, 2-oxoglutarate and fructose. 4. Angiotensin II slightly, but significantly, increased gluconeogenesis from lactate. 5. Gluconeogenesis from glycerol as sole substrate was negligible. Gluconeogenesis from combinations of glycerol together with either lactate, pyruvate, 2-oxoglutarate or malate was appreciably greater than the sum of the glucose output observed when these substrates were added singly.
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PMID:Gluconeogenesis in guinea pig renal tubule fragments--effects of noradrenaline, 3':5' cyclic AMP and angiotensin II. 613 91

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.
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PMID:Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes. 626 35

Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.
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PMID:Renal glycerol metabolism and the distribution of glycerol kinase in rabbit nephron. 627 52

Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.
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PMID:A high yield method for isolating rat islets of Langerhans using differential sensitivity to freezing. 630 93

When skeletal, cardiac, and smooth muscle is exhaustively extracted with a protein-unfolding reagent such as 6 M guanidine HCl and a disulfide-reducing reagent such as 5% beta-mercaptoethanol, a tissue ghost remains intact and retains the characteristic shape and dimensions of the tissue before extraction. In the case of chicken pectoral muscle, the tissue ghost contains 1% of the original muscle proteins. Guanidine HCl extraction followed by collagenase treatment of glycerol-extracted chicken pectoral muscle releases a clean preparation of elongated structures containing 0.2% of the original protein and representing the covalently cross-linked remnants of the muscle fibers. The material of these muscle fiber ghosts extends throughout the interior of the cell. Antibodies raised against the tissue ghosts of smooth muscle cross-react with glycerol extracted skeletal myofibrils, forming a banding pattern which coincides with the banding pattern observed when myofibrils are reacted with antibodies against titin. Titin, a large and soluble protein found in skeletal muscle, cross-reacts with our antigizzard antibody. However, amino acid analysis of the muscle fiber ghosts indicates that titin cannot be the only subunit of the insoluble polymer, but that one or more proteins with a very high glycine and alanine content and a very low basic and acidic amino acid content must also form part of the covalently cross-linked matrix. The possibility is presented that this matrix may be the basis of the superthin 2-3-nm filaments which have been observed in a variety of cell types.
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PMID:A covalently cross-linked matrix in skeletal muscle fibers. 636 89

Our study describes a useful procedure for cryopreservation of pancreatic islet cells. The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment. The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol. One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C). Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1%. This rate was comparable to those of several tissue culture cell lines frozen under similar conditions. Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture. Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours. The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.
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PMID:Cryopreservation of pancreatic islet cells. 637 Nov 65

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.
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PMID:Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide. 640 52

Adipose tissue derived from open biopsies was used to develop a system for studying insulin resistance in human tissue in vitro. Subcutaneous adipose tissue obtained from obese donors was incubated in Parker's medium 199 in the absence or presence of insulin for 24 h under sterile conditions. Adipocytes were then isolated by collagenase digestion, washed thoroughly, and incubated for 2 h with multiple insulin concentrations in Krebs-Ringer phosphate buffer with 4% bovine serum albumin. Lipolysis was estimated by measuring glycerol. Basal lipolysis in adipocytes cultured with insulin did not differ significantly from that of adipocytes cultured without insulin (2.49 +/- 0.18 vs. 2.67+/- 0.58 mumol glycerol/mmol triglyceride). The maximum acute response in adipocytes prepared from tissue exposed to insulin during culture was 55% inhibition of basal lipolysis, whereas the maximum response in cells prepared from tissue not exposed to insulin chronically was 80%. Statistical analysis by paired t test showed a significant difference (P < 0.01) in the reaction of the two groups of cells to acute exposure to insulin. The insulin dose required to produce the half-maximal effect was increased from 3 to 24 microU/ml. Thus, after chronic exposure to insulin, adipocytes were not as responsive to the acute antilipolytic action of the hormone. We conclude that chronic exposure to insulin induces insulin resistance in human adipocytes.
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PMID:Insulin-induced insulin resistance of lipolysis in human adipocytes in organ culture. 699 2

A rapid method for preparing Leydig cells from rat testes is described. An interstitial cell suspension, prepared by collagenase treatment of decapsulated testes, was centrifugal for 10 min over a cushion of 60% (v/v) Percoll to remove red blood cells, and then centrifuged for 20 min in a 0-60% linear density gradient of Percoll. Seventy-four per cent of the cells present in that fraction of the gradient comprising 35-50% Percoll were Leydig cells; the yield from each testis was about 1.5 x 10(6) cells. The Leydig cells appeared viable, excluded Trypan blue, possessed high-affinity binding sites for human chorionic gonadotrophin (hCG) and synthesized increased quantities of testosterone in response to hCG. The cells could be stored overnight in 20% (v/v) glycerol at -20 degrees C, with only minimal effect on the specific activities of a number of enzymes used as markers of subcellular components. Testosterone production in vitro by the cells after storage for 20 h was greater than that of hCG-stimulated fresh cells and was not further increased by hCG.
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PMID:Isolation of rat Leydig cells by density gradient centrifugation. 703 18

There have been numerous reports suggesting the existence of two or more lipases in liver capable of hydrolyzing triacylglycerols at neutral to alkaline pH. We set out to determine if rat liver contains an alkaline triacylglycerol lipase, in addition to heparin-releasable lipase, which has an intracellular localization. We report here the results of studies concerning the pH dependence, subcellular localization and kinetic analysis of the alkaline lipase(s) of rat liver. Homogenates and cytosolic, microsomal and plasma membrane-enriched subfractions all exhibited an optimum of lipase activity at approx. pH 8.0. In no case was there evidence of multiple pH optima in the alkaline ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the com- and subfractions prepared from control livers with those prepared from livers perfused with collagenase. The loss (93%) of lipase activity from both the cytosolic and microsomal subfractions after collagenase perfusion was identical to the loss (93%) of activity from the homogenates, suggesting a common origin with the collagenase-sensitive alkaline lipase on plasma membrane. The characteristics of hydrolysis in vitro of triacylglycerol contained in artificial and natural substrate preparations by the alkaline lipase of rat liver were examined. The artificial substrate preparation was emulsified tri[1-14C]oleoylglycerol prepared by sonication and the natural substrate preparation was a triacylglycerol-rich lipid fraction ('liver fat') prepared from rat liver homogenates. Although the curves were complex, apparent Km values (mean +/- S.W., n = 3-6) over the limited concentration ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the complexity of these kinetics was related to changes in the products of lipolysis, we examined the products after incubations of plasma membrane-enriched fractions with lower and higher concentrations of triacylglycerol. In either case, the products of lipolysis were diacylglycerol, fatty acids and glycerol; no monoacylglycerol accumulated under any circumstances. At the lower concentrations of either tri[1-14C]oleoylglycerol or liver fat, most triacylglycerol hydrolyzed was degraded fully to fatty acids and glycerol. At the higher triacylglycerol concentrations, while complete degradation continued, virtually all of the increased lipolysis of triacylglycerol (over the lipolysis at the lower substrate concentrations) yielded diacylglycerol. The data indicated that the hydrolysis of diacylglycerol by the alkaline lipase of rat liver occurred at a rate slower than that of triacylglycerol. If the same enzyme catalyzes the lipolysis of both tri- and diacylglycerols, triacylglycerols would appear to be preferred...
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PMID:Evidence for the existence of only one triacylglycerol lipase of rat liver active at alkaline pH. 728 28


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