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) Glucose stimulates the incorporation of amino acids into protein in lung cells isolated by digestion of the lung stroma with collagenase. This effect reflects mainly an increase in protein synthesis since no effect of glucose had been found to the uptake of amino acid precursors and, although glucose decreases the rate of intracellular proteolysis by 15%, this effect cannot account for the increased incorporation of radioactivity into proteins. Furthermore, glucose did not induce any significant change in the intracellular content of valine. (2) For glucose to act on protein synthesis, it must be glycolyzed since its stereoisomer, L-glucose, which is not metabolized by lung cells, has no effect. (3) The mechanism of glucose action does not seem to be related simply to variations of cellular ATP content or energy charge. The following arguments seem to support this conclusion: (i) glucose does not bring about significant variations in the concentration of reactants of the adenylate system; (ii) the increase in protein synthesis induced by glucose in energy-depleted cells correlates with a rise in ATP content and energy charge; however, adenosine, which increases ATP levels in a form quantitatively similar to glucose, is unable to affect protein synthesis: (iii) glucose also accelerates the incorporation of amino acids into proteins in adenosine-treated lung cells in which the ATP concentration was almost double that of the control and the energy charge was considerably elevated, ruling out the possibility that a rise in the steady-state concentration of ATP and/or energy charge alone could be responsible for the acceleration of protein synthesis. (4) It can be concluded that the effect of glucose in increasing protein synthesis in lung cells is dependent on some signal arising from its breakdown and not to variations in the concentration of reactants or energy charge of the adenylate system.
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PMID:Role of the adenylate system and glycolytic flux in the control of protein synthesis in isolated rat lung cells. 629 28

We have used cultured trypsin-collagenase-dispersed cells from uteri of 21-day-old rats to investigate the mechanism of control of uterine motility by the beta-adrenergic receptor. After 5 to 7 days in RPMI 1640 the cells started to assume some of the morphological characteristics of smooth muscle cells. When cultures were incubated with 45Ca2+ for 3 h then washed free of isotope and incubated in medium with unlabeled Ca2+, efflux from the prelabeled intracellular pools was linear for up to 60 min. The potent beta-adrenergic agonist isoproterenol had a rapid effect on the rate of efflux and increased it almost sevenfold. Isoproterenol's effect was blocked by propranolol and could be duplicated by the addition of 8-bromo-adenosine 3',5'-cyclic monophosphate or cholera toxin. The cultured myometrial cells had adenylate cyclase properties similar to those of intact muscle strips when these were determined by the conversion of radioactive substrate (alpha-32P-ATP) to 32P-cAMP using a broken-cell preparation. Adenylate cyclase was sensitive to stimulation by GTP and by isoproterenol in the presence but not in the absence of GTP. Adenylate cyclase was also sensitive to stimulation by Ca2+ in the absence of GTP. We conclude that the primary cultures had the properties expected of smooth muscle cells including beta-adrenergic receptors that were coupled to a physiologically important function, Ca2+ flux. The beta-adrenergic receptor's effect on Ca2+ flux was cAMP mediated, and the divalent cation may also regulate its rate of flux by an effect on Ca2+-sensitive cAMP production.
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PMID:beta-Adrenergic catecholamine-dependent properties of rat myometrium primary cultures. 630 58

The incorporation of methionine, lysine, and leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of C-6 glial tumor cells in minimal medium. Although incorporation proceeded at linear rates in both preparations for more than 1 h and into the same spectrum of proteins, Ca2+-restored cells incorporated amino acid 5- to 10-fold more rapidly than Ca2+-depleted cells. Addition of approximately 200 microM Ca2+ in excess of chelator was required to achieve maximal rates of incorporation in Ca2+-depleted preparations. Stimulation by Ca2+ was rapid in onset (several minutes) and slowly reversible by chelator. Ca2+ was uniquely potent and specific among physiologically occurring cations in conferring such stimulation. Stimulation of amino acid incorporation by Ca2+ occurred over a broad range of pH and osmolarities and was facilitated by Mg2+. The effects of Ca2+ in stimulating amino acid incorporation were not traceable to changes in cAMP metabolism, amino acid uptake, protein catabolism, cell ATP or GTP content, or aminoacylation of transfer RNA. Actinomycin D (1 microgram/ml) did not block the stimulatory effects of Ca2+ although puromycin and cycloheximide did. The stimulatory effects of Ca2+ on protein synthesis were not restricted to C-6 in minimal medium. Protein synthesis was reduced by ethylene glycol bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid 40 to 75% in C-6 glioma, GH3 pituitary tumor, PC-12 adrenal tumor, N2A neuroblastoma, and HeLa cells incubated under simulated growth conditions with various enriched media and sera. Ca2+-depleted S49 lymphoma, CHO ovarian tumor, and normal, dispersed chicken embryo cells in enriched medium responded to Ca2+ restoration with increased rates of protein synthesis as did collagenase-dispersed normal rat liver cells in minimal medium. Protein synthesis in rabbit reticulocyte lysates was also inhibited by Ca2+-selective chelators or by Ca2+ removal by parvalbumin affinity chromatography and the inhibition was reversed by Ca2+. These findings are consistent with the existence of a Ca2+ requirement in the translational phase of protein synthesis in eukaryotic cells.
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PMID:Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells. 631 27

HCO-3 transport (JHCO-3) in early juxtamedullary proximal convoluted tubules isolated from infant rabbits during the 1st 3 wk of life is about one-third that in tubules obtained from adults. A rapid increase in transport ensues during wk 4 through 6, so that near-mature levels are attained by the end of this time. Because the pattern for development of glucose absorption was similar and because both HCO-3 and glucose absorption are driven by the lumen-to-cell Na+ flux, the activity of Na-K-ATPase (the Na+-extruding pump) was considered to be a critical mediator. A kinetic microassay (which couples ATP hydrolysis to NADH oxidation) allowed the measurement of Na-K-ATPase and ouabain-insensitive ATPase on the same tubular segment. Three to nine early juxtamedullary proximal convoluted tubules were obtained after collagenase treatment of the kidney and four to six rabbits were studied at each week of life. The mean activity of Na-K-ATPase during the 1st wk of life was 44.5 +/- 3.5 pmol X min-1 X mm-1, one-third of the adult level. During an interim period of development (2-6 wk), enzyme activity gradually reached 60% of adult levels (76.3 +/- 3.0 at 6 wk), while transport of HCO-3 and glucose, studied previously in other animals, attained mature rates. Only in the 7th wk did the enzyme activity reach that of the adult (106.8 +/- 6.8 in wk 7 vs. 128.4 +/- 14.0 in adult rabbits).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Development of solute transport in rabbit proximal tubule. III. Na-K-ATPase activity. 633 Nov 74

To determine the number of Na-K-ATPase units and the enzyme's turnover rate along the rabbit nephron, the specific binding of [3H]ouabain and the Na-K-ATPase activity were measured in single nephron segments microdissected from collagenase-treated kidneys. The highest density of Na-K-ATPase (20-30 fmol X mm-1) was found in the distal convoluted tubule and the medullary thick ascending limb. Binding was intermediate (10 fmol X mm-1) in the proximal convoluted tubule and connecting tubule, and it was lowest (2-7 fmol X mm-1) in the pars recta, the cortical thick ascending limb, and the collecting tubule. In the medullary thick ascending limb, Scatchard analysis of the specific [3H]ouabain binding indicated a dissociation constant of 1.8 microM. The pump activity was proportional to the number of catalytic units, indicating that the maximal turnover rate of Na-K-ATPase (2,000 ATP molecules per minute per ouabain binding site) was similar in the various segments of the nephron. The method developed for quantitating [3H]ouabain binding is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine the number of Na-K-ATPase units in each region of the nephron.
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PMID:Quantitation of [3H]ouabain binding and turnover of Na-K-ATPase along the rabbit nephron. 633 Dec

Properties of isolated single smooth muscle cells in which the surface membrane has been made highly permeable by saponin treatment are described. The single cells were isolated from guinea pig taenia caeci by digestion with collagenase and skinned in a relaxing solution by treatment with 7 micrograms/ml saponin for 10 min. The skinned single cells showed Ca2+-regulated shortening in the presence of Mg-ATP, and the maximum degree of shortening was larger than that of the potassium-induced shortening observed in the intact isolated single cells. The half maximum shortening of the skinned single cells occurred at about 5 x 10(-8) Ca2+. The skinned single cells showed a significantly higher Ca2+-sensitivity than the skinned fiber bundles. The shortening-pCa curve for the skinned single cells was unchanged by alteration of pH and ionic strength, but it was shifted to the left by increasing MgATP concentration or to the right by increasing free Mg2+ concentration. The skinned single cells retained their internal Ca2+ storage site function. Caffeine induced shortening in the skinned single cells preloaded with Ca2+, and this shortening was suppressed by procaine. The release of Ca2+ from the storage site could be produced and facilitated by Ca2+ itself when the skinned single cells were exposed to Ca2+ with a concentration of about 2 x 10(-8) M and this release was suppressed by procaine. These results suggest that the Ca2+-induced Ca2+ release mechanism may play a role in the regulation of the stored Ca2+ in this cell.
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PMID:Some properties of chemically skinned single smooth muscle cells. 633 46

A procedure for the isolation of myocytes from adult rat hearts is described. It is based on successive treatments with Ca2+-free medium, disaggregating enzymes (collagenase and hyaluronidase) and mechanical agitation. Several recent isolation methods were compared and their best features were combined, together with some original modifications. A good yield of high purity myocytes with excellent morphological and functional integrity was obtained. The cells are tolerant to physiological concentrations of Ca2+. Cellular levels of ATP, Na+, and K+ are close to those in intact hearts and glucose oxidation rates and succinate exclusion are also close to normal. These characteristics are maintained for periods over 1 h.
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PMID:Isolation of Ca2+-tolerant myocytes from adult rat heart. 642 25

ATP content in a nerve cell isolated from dorsal root ganglia of adult guinea-pigs by collagenase was measured by a newly developed technique modified from the conventional luciferin-luciferase methods. A small volume (4 microliters) of the nerve cell suspension, which contained 10-300 nerve cells (3-100 X 10(-4) microliters of cellular volume) under view of an inverted, phase-contrast microscope, was heat-treated for about 1 s by flame of an alcohol lamp. This heat-treated cell suspension was then reacted with a luciferin-luciferase solution. Light flux from the bioluminescence thus elicited gave an ATP content in single nerve cell, 27 pg (mean) +/- 10 pg (S.D.). ATP concentration in a nerve cell was calculated as 1.7 mM (mean) +/- 0.6 mM. The ATP content in a nerve cell was reduced when the nerve cells were exposed to KCN (5 microM) or dinitrophenol (20 microM), respectively.
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PMID:ATP content in isolated mammalian nerve cells assayed by a modified luciferin-luciferase method. 662 Oct 97

High yields of Ca2+ - stable myocytes were obtained by perfusion of adult rat heart with a buffered collagenase medium followed by mincing and three additional digestion periods. Release of lactate dehydrogenase, respiratory control, content of ATP and creatine phosphate, electrical stimulation and attachment to extracellular matrix components indicated that the sarcolemma of the isolated myocytes remained intact and that the cells maintained some of the most basic physiological functions. The myocytes maintained their rod-shape in a medium containing 2.5 mM of Ca2+ and their release of LDH was slow. Some of the myocytes were contracting spontaneously, at a low rate, in an abrupt end-to-end contraction. Other cells appeared quiescent but they were all able to respond to external electrical stimulus. The oxygen consumption was measured by a perifusion method. In different preparations the basal consumption was 14-26 nmol O2/min X 10(5) rod-shaped myocytes. Freshly isolated rod-shaped heart cells attached in 30 minutes to dishes coated with collagen type IV, laminin or fibronectin but did not attach to dishes coated with collagen type I or III or to collagen gels. Attachment occurred at the ends of the cells.
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PMID:Isolation, characterization and adhesion of calcium-tolerant myocytes from the adult rat heart. 672 24

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.
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PMID:Structural glycoproteins from rabbit aortic media. 687 Aug 24


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