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)

A simple method to determine adenylate cyclase activity in isolated single nephron segments is described. Segments of the proximal convoluted tubule or the cortical collecting tubule were isolated from rabbit kidney slices pretreated with collagenase. After the tubule membranes were made permeable by adding hypotonic medium and freezing-thawing, each sample was incubated at 30 degrees C for 30 min in a medium containing ATP and theophylline. Generated cAMP was succinylated and served for radioimmunoassay. Addition of the incubation medium did not interfere the radioimmunoassay. Recovery of added cAMP was 96%. In the proximal convoluted tubule, either 8 mM NaF or 1 U/ml parathyroid hormone (PTH) markedly stimulated adenylate cyclase activity, but 1 mU/ml arginine vasopressin (AVP) did not. By contrast, in the cortical collecting tubule, either 8 mM NaF or 1 mU/ML AVP markedly stimulated adenylate cyclase activity, but 1 U/ml PTH did not. These data imply that this method is sensitive enough to detect either specific or nonspecific response of adenylate cyclase activity in single nephron segments.
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PMID:A simple method to determine adenylate cyclase activity in isolated single nephron segments by radioimmunoassay for succinyl adenosine 3',5'-cyclic monophosphate. 22 39

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.
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PMID:Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes. 31 46

The extraction of isolated vertebrate smooth muscle cells at high and low ionic strength yields cell ghosts which are seen in the electron microscope to be composed of a complex network of 10-nm filaments, together with residual actin. After SDS-gel electrophoresis of the cell ghosts only 2 bands may be recognized, one corresponding to actin and the other migrating at about 55 000 mol. wt that arises from the 10-nm filaments. The 10-nm filaments are extremely sensitive to proteolysis and are absent from cells exposed to crude collagenase in the presence of Triton X-100. Such cells, lacking 10-nm filaments, still contract in response to ATP. The data indicate that the 10-nm filaments are not essential for contraction, but rather form a specialized intracellular cytoskeleton. While completely insoluble in concentrated salt solutions the 55 000 mol. wt protein is readily extracted with acetic acid from homogenized and salt-extracted smooth muscle residue. The extracted protein reassembles, on dialysis, into filaments of about 10-nm diameter and has an amino acid composition almost identical to that deduced for vertebrate neurofilaments. From the cytoskeletal role that the 10-nm filaments play in smooth muscle and, as appears likely, in other cell types the filament protein has been tentatively termed 'skeletin'. Results relating to the proportion of skeletin in smooth muscle and the structure of the 10-nm filaments are described and discussed.
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PMID:Studies on the function and composition of the 10-NM(100-A) filaments of vertebrate smooth muscle. 56 Oct 84

1. A method for the preparation of isolated mammary gland cells of the rat is described. 2. The procedure involves disaggregation of the tissue in a collagenase-hyaluronidase mixture and subsequent purification of the heterogeneous population of cells by centrifugation in discontinuous Ficoll-400 gradients; the preparation takes 60 minutes. The yield of cells is approximately 14%. 3. The cells as prepared have high rates of metabolism and synthetic capacity and exhibit metabolic characteristics comparable to intact tissue. 4. Measurements of the content of metabolic intermediates show cells to have, and retain, outstandingly high levels of ATP and to have an energy charge close to 0.9. Levels of other intermediates approximate to those found in the intact tissue. The level of glycolytic intermediates below the triose phosphate stage indicate the highly aerobic state of the cells. 5. The pattern and scale of glucose utilization, measured using specifically labelled glucose incorporation into 14CO2 and 14C-labelled lipid production, approximates closely in isolated cells at 5 and 20 mM glucose and in tissue slices at 20 mM glucose concentration. Mammary gland slices incubated with 5 mM glucose have a considerably lower rate of metabolism. Isolated cells exhibit a higher proportionate rate of glucose utilization by way of the pentose phosphate pathway. 6. The isolated cells are hormone responsive. Insulin increases the oxidation of glucose by the pentose phosphate pathway and stimulates lipid synthesis. Addition of progesterone and cortisone in vitro (10 muM) leads to a marked and rapid decrease in the rate of glucose oxidation and conversion to lipid.
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PMID:Regulation of mammary gland metabolism: pathways of glucose utilization, metabolite profile and hormone response of a modified mammary gland cell preparation. 67 49

1. A simple procedure for the isolation of morphologically intact, metabolically viable sheep liver parenchymal cells is described in detail. 2. The method is based on the initial treatment of fresh liver slices with collagenase and hyaluronidase. 3. The cell preparation was studied with respect to membrane permeability, potassium content, ATP/ADP ratio, adenylate content, and gluconeogenic capacity with respect to various substrates. 4. Data are present with respect to the distribution of phosphoenolpyruvate carboxykinase in isolated cells and whole sheep liver. 5. The results are compared, where possible, with data currently available from isolated perfused sheep liver and multi-catheterised animals.
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PMID:Preparation and biochemical characterisation of isolated parenchymal cells from adult sheep liver. 83 5

The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubationof the cells, as judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested as an additional criterion of cell function. Adenine nucleotides synthesized from [14C]adenine were slowly catabolized to allantoin, with little incorporationof radioactivity into other purine compounds. [14C]Adenine is thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthine were rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosine was initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has important functions for the whole organism.
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PMID:Purine metabolism in isolated rat hepatocytes. 92 48

Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes.
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PMID:Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation. 95 71

Rat hepatocytes were isolated by liver perfusion in the presence of collagenase and hyaluronidase and incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, noted as an increase in succinate-stimulated oxygen uptake, a decrease in trypan blue exclusion frequency, a leakage of cytosolic lactate dehydrogenase activity and an increased proportion of swollen and disrupted cells. After anaerobic incubation for 30 minutes--but not for 60 minutes--there were signs of recovery from anoxic cell injury upon re-oxygenation. The changes in plasma membrane permeability properties in anoxia seemed to be preceded by a marked decrease in cellular ATP level; aerobic incubation of hepatocytes in the presence of an uncoupler of phosphorylation from respiration led to a similar decrease in cellular ATP concentration followed by similar disturbances in plasma membrane permeability properties. It is suggested that a distrubed plasma membrane function caused by a decreased energy level is of primary importance for the initiation of cell death in anoxia.
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PMID:Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia. 100 75

The adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] sensitivity to salmon calcitonin in 11 different segments of the rabbit nephron was investigated using a micromethod for enzyme activity measurements in samples, each containing a single piece of tubule. The required segments were isolated by microdissection from collagenase-treated rabbit kidneys. The results were expressed as femtomoles of adenosine 3':5'-cyclic monophosphate formed per mm of tubular length per 30 min of incubation time. In the presence of 0.1 mug/ml of synthetic salmon calcitonin, it was found that eight segments exhibited no hormonal sensitivity whereas maximal responses were induced in three segments, the "bright" portion of the distal convoluted tubule, the cortical and the medullary portions of the thick ascending limb of the loop of Henle (stimulated over control activity ratios were 32, 11, and27). The very high sensitivity to calcitonin of the adenylate cyclase contained in these three segments (0.01 ng/ml of salmon calcitonin inducing a 2-fold stimulation; half-miximal stimulation corresponding to about 0.3 ng/ml of salmon calcitonin) suggests that the distal convoluted tubule, as well as th cortical and medullary portions of the thick ascending limb of the loop of Henle represent physiological target structures of calcitonin action within the kidney.
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PMID:Distribution of calcitonin-sensitive adenylate cyclase activity along the rabbit kidney tubule. 106 74

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.
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PMID:Relationship of energy production to gluconeogenesis in renal cortical tubules. 117 38


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