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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the dystrophic influence on the characteristics of actin, a method for the isolation of F-actin filaments from the skeletal muscle of small sizes, i.e., less than 0.5 g, was devised. In this method, minced muscle was treated with collagenase and hyaluronidase, and the isolated filaments were washed with adenosine triphosphate (ATP). Upon examination in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the ATP-washed filaments showed a protein component identical in mobility to actin in untreated myofibrils or to that prepared by the conventional method. Electron microscopic appearances of the filaments were similar to those of F-actin filaments described in the literature. The dimensions of the filaments were 0.5--2.5 micrometer in length and 60--70 A in diameter. The ability to activate the Mg-adenosine triphosphatase or myosin was found to be Ca2+ independent. In all aspects of the above characteristics, the filaments from leg muscles of 129/Re dydy dystrophic mice and their litter mates were observed to be identical.
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PMID:Isolation of F-actin filaments. Comparison of F-actin filament preparations from normal and dystrophic mouse muscle. 15 92

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.
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PMID:Analytic subcellular fractionation of acini from rat lacrimal gland. 217 90

Cells isolated by hyaluronidase incubation from chicken small intestine were used to study the effects of anisosmotic buffers on K+ transport. Hypo-osmolarity (200 mosmol.l-1) reduced both the ouabain-sensitive and the ouabain-resistant, but bumetanide-sensitive, net K+ influx and increased the K+ efflux. The hypo-osmolarity induced K+ efflux was prevented by quinine and unaffected by bumetanide. These results suggest that Ca2+-activated K+ channels may be involved in regulatory volume decrease in chicken enterocytes. Hyperosmotic conditions (400 mosmol.l-1) increased the portion of net K+ influx mediated by the Na+/K+-ATPase and that mediated by the bumetanide-sensitive K+ transport system, and decreased the K+ efflux.
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PMID:Effects of anisosmotic buffers on K+ transport in isolated chicken enterocytes. 253 56

Using the incubated isolated rat retina, the effects of hyaluronidase on the electroretinogram (ERG) and metabolic activities were investigated. Initial experiments established the activity of hyaluronidase needed to liquefy, within 15 to 30 minutes, the vitreous of postmortem human eyes; this concentration was 1,000 units/mL. Rat retinas were superfused with a bicarbonate-buffered, oxygenated medium to which hyaluronidase was added in activities ranging from 100 to 5,000 units/mL. These concentrations of hyaluronidase did not significantly alter the amplitudes of the a waves and b waves of the ERG in comparison to their control amplitudes. Measurements were also made of lactic acid production, oxygen consumption, glutathione content, and adenosine triphosphatase activities in control and hyaluronidase-exposed retinas. In the presence of hyaluronidase, their respective values were similar to the controls for all biochemical factors studied. The present experiments demonstrate that addition of hyaluronidase to an "ocular irrigating" solution results in normal ERGs and normal retinal metabolic activity and suggests the possibility that hyaluronidase may be useful in enzyme-assisted vitrectomy.
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PMID:Hyaluronidase and retinal function. 293 18

Rat renal papillary collecting duct (PCD) cells were isolated using collagenase and hyaluronidase digestion and a three-step low-speed centrifugation. As assessed by binding of the lectin Dolichos biflorus and determination of vasopressin-sensitive adenylate cyclase and Na+-K+-ATPase, the enrichment of PCD cells over a crude papillary cell preparation was 1.8, 2.4, and 1.4, respectively. Microscopic evaluation indicated that the preparation was greater than 90% pure PCD cells. The isolated cells were viable as evident from the high K/Na ratio of intracellular electrolytes measured by electron probe analysis (5.3), from the high ATP/ADP ratio (2.15), and the metabolic response to alterations in Na transport. Exposure to 2 mM ouabain or removal of Na reduced O2 consumption by 25-35%; the uncoupler carboxylcyanide-m-chlorophenylhydrazone more than doubled O2 consumption. In the presence of 14 mM glucose and at a PO2 of 100 Torr the cells produced substantial quantities of lactate. This aerobic glycolysis may account for greater than 20% of the ATP production. In the presence of rotenone, glycolysis increased by 56% and was able to maintain the cellular ATP level at 65% of control. In the absence of any exogenous substrate PCD cells respired normally and had a close to normal ATP content, but lactate production was markedly decreased. These results demonstrate that viable PCD cells can be isolated from rat kidney. At normal PO2 and in the presence of D-glucose the cells show a substantial amount of aerobic glycolysis, although their mitochondrial respiration is not rate limiting. In the absence of glucose the cells derive the majority of their energy from an as yet unidentified endogenous substrate.
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PMID:Purification of rat papillary collecting duct cells: functional and metabolic assessment. 330 74

Isolated acini from lactating mouse mammary glands were prepared by collagenase and hyaluronidase digestion of tissue. Mammary tissue or acini incubated in vitro in tissue culture medium or a similar Ringer's solution lost K and gained Na. Intracellular concentrations approached, but did not equal, the concentrations in the external solution. This ion shift was largely prevented by incubating in a solution with ionic composition resembling mouse milk. In paired experiments, incubation with ouabain (1 mM) caused further increases in Na and decrease in K, suggesting that a functional Na+-K+-ATPase was present. Viability of acini was indicated by normal ATP content and morphology. The ion shift in NaCl-based solutions was slower at 0 degrees C than at 37 degrees C, suggesting that the flux is a membrane-regulated process. Under identical procedures, ion shifts did not occur in thymocytes or a cultured mammary cell line but were seen in both lactating and nonlactating mammary tissue. Nonlactating mammary tissue had a high Na and low K concentration in vivo. As predicted by previous models for the mechanisms of milk secretion, intracellular electrolyte content in mammary epithelial cells appears to be responsive to the ion concentration in the extracellular environment.
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PMID:Sodium and potassium content and viability of mouse mammary gland tissue and acini. 337 13

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.
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PMID:Changes in the lipid content of boar sperm plasma membranes during epididymal maturation. 399 37

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.
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PMID:Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. 625 27

Calcium-tolerant myocytes were isolated from adult rat ventricles by successive perfusion and incubation with buffer containing collagenase and hyaluronidase. Greater than 70% of the cells excluded trypan blue, maintained normal morphology, and contracted in response to an externally applied electric field. We have characterized metabolic defects present in isolated calcium-tolerance myocytes when exposed to low concentrations of extracellular calcium under aerobic and anaerobic conditions. In control cells exposed to 1.25 mM Ca2+, the following metabolic parameters were measured (in mumol/g protein): adenosine triphosphate (ATP) 28.8 +/- 3.3, creatine phosphate (CrP) 49.1 +/- 7.5, intracellular Na+ 37.7 +/- 8.1, intracellular K+ 352.9 +/- 49.3, cellular Ca2+ 12.3 +/- 1.8, as well as rate of protein synthesis 0.34 +/- 0.03 mumol . g protein-1 . h-1. In aerobic cells incubated in medium without added Ca2+, the corresponding values (in mumol/g protein) were ATP 27.9 +/- 4.4, CrP 25.3 +/- 4.3, intracellular Na+ 130.9 +/- 23.1, intracellular K+ 217.2 +/- 32.0, cellular Ca2+ 3.9 +/- 1.0, and rate of protein synthesis 0.09 +/- 0.02 mumol . g protein-1 . h-1. These data indicated major metabolic aberrations in myocytes exposed to medium low in Ca2+ (less than 10 microM). Metabolic depression was most severe in cells incubated in the absence of both Ca2+ and O2. It is postulated that Ca2+ removal resulted in an increase in Na+ and K+ permeability, causing a net gain of intracellular Na+ and loss of intracellular K+. These ionic shifts might stimulate the activity of membrane-associated Na+-K+-ATPase, accounting for lower levels of CrP.
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PMID:Effects of extracellular calcium removal and anoxia on isolated rat myocytes. 711 49

In preparation for pulse-chase autoradiography experiments and studies of cell surface changes of relevance to plasma membrane biogenesis, we have prepared a cell suspension from the salt gland of ducklings. The method used was a modification of previous methods used for pancreas and salivary gland and included digestions with collagenase and hyaluronidase, divalent cation chelation, and dispersion by gentle pipetting. Yields were 1.13 X 10(7) cells/g gland, and cell recovery was 45% by DNA assay. Recovery of Na,K-ATPase, a marker for salt gland secretory cells was 40--47%. Cell viability was strongly indicated by trypan blue exclusion and 3H-leucine incorporation. Transmission and scanning electron microscopy revealed that most cells retained ultrastructural features characteristic of the intact gland. Smaller cells (3--8 micrometers in diameter), exhibiting few surface microvilli and relatively few cytoplasmic organelles, likely represented the undifferentiated, peripheral cells from the tips of secretory tubules. Larger cells (5--10 micrometers in diameter), exhibiting prominent surface membrane folds enclosing numerous mitochondria, likely represented the functional, secretory cells of the salt gland tubules in various stages of differentiation. The surface folds presented as microvilli and microplicae in scanning electron micrographs.
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PMID:Preparation and characterization of single cells from the avian salt gland. 742 16


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