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 method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.
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PMID:Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments. 16 67

An enzymatic method is described for isolating intact parenchymal cells from rat livers. 3--4 g cells (wet weight) could be isolated from livers of rats weighing 180--230 g. After an in vitro preperfusion of 15 minutes with a Ca-free buffer, collagenase (200 mg/1) and calcium chloride (5.2 mmol/1) were added. Perfusion was continued for another 15 minutes at 37 degrees C. Micromorphological integrity of cell membranes was demonstrated by scanning electron microscopy. With regard to rates of gluconeogenesis and protein synthesis, parenchymal cells isolated according to our method were found to be superior to liver slices and cells isolated by other methods. Ratios of ATP/ADP (5.69) and of lactate/pyruvate (8.64) as parameters of the energetic situation and the redox state resp. were found within the physiological range. Integrity of cell surface receptors was proved by their sensitivity to epinephrine, glucagon and insulin. Glucagon (0.3 mumol/1) and epinephrine (1 mumol/1) and reduced glycogen deposition in hepatocytes of fasted rats by 84.9 % and 95.9 % resp. Both hormones stimulated glycogenolysis in parenchymal cells of fed rats to a similar extent. Urea synthesis was stimulated 29.5 % by glucagon (1 mumol/1), and inhibited 28.0 % by insulin (10 nmol/1). The stimulatory effect of glucagon (1 mumol/1) was abolished by insulin (10 nmol/1).
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PMID:[Isolation of intact liver parenchymal cells by a modified enzymatic method]. 16 41

Adenyl cyclase activity of rat pancreatic islet membrane was increased by secretin, pancreozymin, and isoproterenol, while ACTH, glucagon, growth hormone, and insulin had no effect. Both secretin and isoproterenol activations were enhanced by prostaglandin E1 (PGE1) and GTP. Isoproterenol activation was additive with PGE1, as was that of secretin with PGE1, but only in the presence of GTP. Secretin activation in the presence of PGE1 and GTP was equivalent to NaF stimulation. Kinetic analysis indicated that secretin and GTP increased the maximum velocity of the adenyl cyclase and tended to decrease the apparent affinity of the enzyme for ATP. Glucagon activation of islet membrane adenyl cyclase was dependent upon prior treatment of the membrane preparation with EGTA and the use of inhibitors of proteolytic enzymes during the collagenase digestion phase of islet preparation. These results suggest that hormonal regulation of insulin secretion may be affected by PGE1 and guanine nucleotide modulation of the adenyl cyclase activation process.
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PMID:Hormonal regulation of pancreatic islet adenyl cyclase. 17 51

Hepatocytes isolated from the liver of the common goldfish Carassius auratus L. with crude bacterial collagenase maintained ATP levels for at least 2 h. Glycogenolysis was maximally activated by 1 X 10(-6) M epinephrine and 5.8 X 10(-9) M glucagon. In liver cells incubated in calcium-free buffer containing 1 mM ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, basal glycogenolysis was enhanced by the addition of 1-4 mM calcium but the elevation of cyclic AMP and glycogenolysis due to epinephrine was unaffected by calcium. The divalent cation ionophore A23187 did not alter basal or hormone-stimulated glycogenolysis. Isoproterenol was approximately as potent as epinephrine but phenylephrine was glycogenolytic only at very high concentrations. l-Propranolol competitively inhibited the increased glycogenolysis due to catecholamines but phentolamine was ineffective as a blocking agent. Isoproterenol and epinephrine stimulated glycogenolysis at lower concentrations than those required to elevate cyclic AMP accumulation. Phenylephrine was without effect on cyclic AMP. Propranolol competitively inhibited both epinephrine- and isoproterenol-stimulated cyclic AMP accumulation, but phentolamine did not block either response. Catecholamine-stimulated glycogenolysis in goldfish liver is apparently a beta-adrenergic effect. However, low concentrations of epinephrine enhance glycogenolysis without affecting total cyclic AMP.
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PMID:Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes. 18 9

The cholinergic sensitivity of rat diaphragm muscle, me-sured as the magnitude of depolarization responses to repetitive, iontophoretic pulses of acetylcholine (ACh) onto neuromuscular endplates, is increased by addition of ATP to the perfusion medium. Depolarization responses begin to increase within the first min after addition of 10 mM ATP and plateau at 60% above control levels (mean value) after 4 to 6 min. Neither the magnitude nor the time course of the potentiations corresponds to changes in resting potential or membrane resistance. Other nucleotides are equally or less effective at the same concentration: ATP=ADP greater than UTP greater than AMP=GTP (=no added nucleotide control) The duration of the individual ACh responses does not increase during continuous exposure to the active nucleotides for up to 15 min except when the muscle is pretreated with eserine. Mild enzymatic predigestion of the muscle with collagenase and then protease, increasing the availability of the postjunctional membrane to bath-applied drugs, decreases the variability and increases the magnitude of the potentiation to a given dose of ATP. The dose-response curve for ATP is then more than half-maximal at 1 mM and the ranking of the other nucleotides relative to ATP is the same as without predigestion. There is an optimum Ca++ concentration for the potentiation between zero and 2 mM: potentiation is enhanced in Ca++ -free medium, partially blocked in twice-normal Ca++ medium, and totally blocked in Ca++ -free medium 10 min after a 5 min exposure to 2.5 mM EGTA. The similar Ca++ dependence of ACh receptor activation in the absence of added nucleotide suggests that ATP directly facilitates receptor activation by ACh. This facilitory action could be one of the physiological roles for the ATP released from stimulated phrenic nerve.
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PMID:Potentiation of postjunctional cholinergic sensitivity of rat diaphragm muscle by high-energy-phosphate adenine nucleotides. 18 89

The hypotheses were tested that the relaxant effect of adenosine and related compounds in the longitudinal muscle of the rabbit small intestine involves interaction with adenylate cyclase and/or the elevation of tissue cAMP levels. Adenylate cyclase was prepared by gentle homogenization of an isolated smooth muscle cell fraction obtained after collagenase digestion of longitudinal muscle strips. A number of analogs and derivatives of adenosine possessing a primary or secondary 6-amino group were found to inhibit the enzyme similarly to adenosine; however, there was no correlation between compounds known to relax the intact tissue and the existence, or the degree of, cyclase inhibition. Isolated muscle strips were exposed to adrenaline, isoprenaline, adenosine or ATP, at doses causing 30-60% relaxation, for 60 sec prior to sampling and analysis of cAMP content. While small increments in cAMP levels were found after administering adrenaline or isoprenaline, no change was found with adenosine in the absence or presence of theophylline of 1-methyl-3-isobutylxanthine. Neither adenylate cyclase inhibition nor changes in cAMP levels appear to be part of the mechanism of the smooth muscle relaxant action of adenosine or ATP.
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PMID:Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle. 18 64

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).
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PMID:Calcium metabolism and amylase release in rat parotid acinar cells. 18 53

Isolated pancreatic acini were prepared by a new method from mouse and rat pancreases by digestion with purified collagenase and chymotrypsin followed by mechanical shearing. Acini were structurally similar to those of the intact pancreas, having a normal luminal structure but with the basal acinar cell membranes exposed to the incubation medium. Amylase release in response to both cholinergic analogues and the cholecystokinin analogues caerulein and pentagastrin was comparable to that of the intact pancreas, but was much greater than previously reported for isolated acinar cells. Cholinergic-stimulated release was inhibited by atropine with a Ki value of 1.4 nM which is comparable to other muscarinic receptors. All agonists tested, when added at supramaximal concentrations, produced a submaximal release of amylase even though ATP levels and the release of slowly exchanging 45Ca2+ were normal or increased. Acini releasing amylase submaximally after being exposed to supramaximal concentrations of carbachol failed to respond to a maximal amount of caerulein or to the Ca2+ ionophore A23187. It is concluded that the decreased response (desensitization) is a postreceptor phenomenon and possibly mediated by Ca2+ itself.
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PMID:Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. 21 42

It was shown previously that addition of cyclic AMP (cAMP) to a synaptic membrane fraction incubated with [gamma-32P]ATP stimulated the phosphorylation of two proteins, designated proteins Ia and Ib, found only in nerve tissue. Addition of Ca2+ plus veratridine to synaptosomes preincubated with 32Pi stimulated the phosphorylation of two proteins with similar apparent molecular weights. Various techniques have now been used to determine whether the two proteins phosphorylated in synaptosomes in the presence of Ca2+ plus veratridine are the same as proteins Ia and Ib phosphorylated in synaptic membranes in the presence of cAMP. The proteins phosphorylated by the two procedures were extracted under similar conditions, had similar apparent molecular weights and charges, and were digested by collagenase at similar rates and to the same radioactive intermediates and end products. Furthermore, the two sets of proteins were digested by three other proteolytic enzymes to phosphopeptides with similar molecular weights. The results indicate that Ca2+ and cAMP are each capable of regulating the phosphorylation of proteins Ia and Ib.
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PMID:Ca2+ and cyclic AMP regulate phosphorylation of same two membrane-associated proteins specific to nerve tissue. 22 28

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.
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PMID:Determination of Na-K-ATPase activity in single segments of the mammalian nephron. 22 55


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