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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

[3-H]Epinephrine binding to isolated purified rat liver plasma membranes is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125mug. Rat liver plasma membranes stored at-70 degrees C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes. Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A2 (phosphatide acylhydrolase EC 3.1.1.4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively. In the presence of 10-3M Mg-2+ ions, increasing concentrations of QTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10-5M GTP, representing an inhibition of 52% of the control. In a Mg-2+ -free system, epinephrine binding was unaffected by GTP. However, in a Mg-2+ -free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10-3 M reduced epinephrine binding to 28% of the control. GRP (10-5 M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane. [3-H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40-200 mug protein). GTP (10-5 M) and ATP (10-4 M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10-2-10-3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [3-H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [3-H]epinephrine binding. Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [3-H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.
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PMID:Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells. 16 9

Goat sperm lysate from cauda epididymis was incubated in the presence of [14C]phosphatidyl-choline, -ethanolamine, -inositol and diphosphatidyl-glycerol. The release of [14C]diacylglycerol from only phosphatidyl inositol confirmed the presence of phosphatidyl inositol specific phospholipase C. The enzyme activity was linear up to 1 hr of incubation at a sperm concentration of 1-10 x 10(9). It had pH optimum of 7.5 in a broad range of pH activity profile (pH 6-9). Maximum activity was observed at 8 mM calcium ion concentration. EDTA and EGTA (5 mM) did not inhibit the activity completely. A comparative study on spermatozoa and fluid from caput, corpus and cauda epididymis revealed 6.5-fold increase of activity in spermatozoa and a 4-fold decrease in case of fluid during the epididymal transit. However, the total protein content remained unchanged in fluid and decreased up to the extent of 2.4-fold in spermatozoa during this process. Thirty five percent of the caudal sperm activity was soluble and the rest was associated with head (44%), mid-piece (10%) and tail (10%).
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PMID:Phosphoinositide specific phospholipase C activity of goat spermatozoa in transit from the caput to the cauda epididymis. 166 Dec 71

The bovine seminal plasma is formed mainly by secretions of epididymis and the glandular epithelia in ampulla, seminal vesicles, prostate and Cowper's glands. The contribution of each organ to the hydrolytic enzyme activities (glycosidases, exopeptidases, phospholipases) of the bull seminal plasma has been analyzed and is reviewed in this paper with special emphasis on the role of the accessory glands. Seminal vesicles seem to have a major role in the secretion of seminal plasma acid alpha-glucosidase, acid alpha-mannosidase and beta-N-acetylhexosaminidase, aminopeptidase A, dipeptidyl peptidase II and IV and gamma-glutamyl transpeptidase as well as Ca(2+)-dependent and Ca(2+)-independent phospholipases A2 with distinct substrate specificities, a choline-specific phospholipase C and a Co2+ (Mn2+)-activated sphingomyelinase. The enzyme pattern in the ampulla closely resembled that of the seminal vesicles and obviously contributes to the seminal plasma level of these hydrolases. The bull prostate and Cowper's glands contained a strong Ca(2+)-dependent phospholipase A2 activity. However, these glands may not contribute to the seminal plasma PLA2 activity. At ejaculation the epididymal spermatozoa are exposed to these enzymes. They may have a specific affinity to sugar, peptide or phospholipid residues at distinct sites of the sperm surface. These enzymes may also participate in the digestion of various other semen components to create a suitable milieu for the emitted spermatozoa.
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PMID:Hydrolases from bovine seminal vesicle, prostate and Cowper's gland. 213 63

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.
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PMID:Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa. 255 6

Insulin is known to increase the de novo synthesis of inositol phospholipids in rat epididymal fat pads. We presently examined the effects of insulin on the hydrolysis of inositol phospholipids in this tissue. Relatively small (30-40%) but significant increases in inositol phosphates (mono-, di-, and tri-) were apparent within 30-60 s of insulin treatment in fat pads (and adipocytes); thereafter, inositol phosphates returned to control levels. These rapid insulin-induced increases in inositol phosphates appeared to be due to phospholipase C-mediated hydrolysis of inositol phospholipids, since there were associated transient decreases in these lipids during 32P pulse-chase experiments. Increases in the synthesis of inositol phospholipids were also apparent within a few minutes of insulin treatment and persisted for at least 2 h. We conclude that, in the rat epididymal fat pad, insulin has two phospholipid effects, viz. a transient activation of phospholipase C, and a persistent increase in de novo phospholipid synthesis.
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PMID:Insulin provokes a transient activation of phospholipase C in the rat epididymal fat pad. 352 74

An enzyme hydrolysing the synthetic substrate p-nitrophenylphosphorylcholine was highly active in the epididymal caput and very low or absent in other parts of the bovine reproductive organs. This enzyme was also found in the secretory fluid obtained from the epididymal caput but much lower activities were encountered in cauda epididymis and seminal plasma. The enzyme displayed an optimum at pH 6.5, an Mr of about 66000, a pI value of 5.0 and was suppressed by chelating agents and reactivated by Co2+ and Zn2+. After partial purification the enzyme also showed hydrolysis of p-nitrophenyl phenylphosphonate, bis-p-nitrophenyl phosphate and hexadecanoyl-p-nitrophenylphosphorylcholine with slightly different pH optima and modifier characteristics. It is concluded that this secretory enzyme is distinct from the membrane-bound phospholipase C and may play a role in processing of the spermatozoan phospholipids during the epididymal maturation.
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PMID:A secretory phospholipase C-like enzyme in the bovine epididymal caput. 372 Sep 58

Epididymal 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal membranes, and using two approaches, we investigated the relationship between 5 alpha-reductase activity and the membrane environment. In the first, nuclear and microsomal membrane fractions were treated with phospholipases to modify specifically the structure of the phospholipid component of the membranes, and the effects of these treatments on the kinetic parameters of 5 alpha-reductase were examined. The second approach was to observe the effects of phospholipids of known structure on solubilized 5 alpha-reductase activity. Treatment of the membrane fractions with phospholipase C increased the Km(app) of both the nuclear and microsomal 5 alpha-reductases for testosterone. Phospholipase A2 treatment also increased the Km(app) of the microsomal enzyme, but in contrast, the Km(app) of the nuclear 5 alpha-reductase for testosterone was unaffected. This demonstrated a fundamental difference in the role of the membrane environment in the expression of 5 alpha-reductase activity in these subcellular compartments. The ability of phospholipids to enhance the activity of solubilized 5 alpha-reductase was highly specific and structure related. Only phosphatidylcholines containing either unsaturated acyl chains or saturated acyl chains of 12 carbon atoms were found to activate 5 alpha-reductase. The most potent activator was dilauroyl phosphatidylcholine, which reduced the Km(app) values of both nuclear and microsomal 5 alpha-reductases for testosterone, without affecting the concentration of active 5 alpha-reductase (Vmax(app) ). This is the first time that an activator of 5 alpha-reductase has been found. These findings suggest that epididymal 5 alpha-reductase activity may be regulated by changes in the phospholipid environment.
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PMID:Modulation of epididymal delta 4-steroid 5 alpha-reductase activity in vitro by the phospholipid environment. 399 84

The change in transmembrane potential of rat adipocytes was measured using the fluorescent probe 3,3'-diethylthiadicarbocyanine iodide, diS-C2-(5). The method was calibrated by altering the potassium ion concentration while keeping the sum of potassium and sodium ions at a constant concentration of 153 mM (Bailey et al: Bioelectrochem. Bioenergetics 21:333-42, 1989). Two insulin-mimetic agents, phospholipase C from Clostridium perfringens and concanavalin A, induced a dose dependent hyperpolarization of rat epididymal adipocytes, like insulin. Removal of endogenous adenosine with adenosine deaminase or adenosine receptor blockade with isobutylmethylxanthine following the initiation of insulin-induced hyperpolarization resulted in depolarization. These same agents induced hyperpolarization of -6 to -8 mV when added without insulin. The replacement of adenosine with its analogue, N6-phenylisopropyladenosine, plus insulin depolarized the cells toward the transmembrane potential established by insulin, -2.0 mV. These studies suggest that adenosine receptor occupancy is required to maintain insulin-induced hyperpolarization.
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PMID:Membrane potential of rat adipocytes: effect of phospholipase C, concanavalin A, and adenosine. 751 7

Recent studies of whole animal responses have defined a role for circulating TGF-beta in the preservation and stabilization of microvascular endothelial function (Lefer et al. [1993] Proc. Natl. Acad. Sci. U.S.A., 90:1018-1022; Pfister et al. [1992] J. Exp. Med., 176:265-269). In order to determine which TGF-beta receptor types are responsible for this endothelial cell responsiveness, we used an affinity-labeling technique with 125I-TGF-beta 1 and -beta 2 to characterize TGF-beta receptors on five different endothelial cell cultures: early passage bovine lung and rat epididymal fat pad microvascular endothelial cells (BLMEC and REEC), established endothelial cell lines from bovine adrenal medulla capillaries (EJG), fetal bovine heart (FBHE), and bovine pulmonary artery (CPAE). Since it is known that endothelial cells from different parts of the vasculature vary with respect to cell surface antigen expression (McCarthy et al. [1991] Trends Pharmacol. Sci., 12:462-467; Augustin et al. [1994] Bioessays, 16:901-906), it is important to compare TGF-beta receptor expression on microvascular and macrovascular endothelial cells. We observed 85 kDa and 200-400 kDa labeled receptor bands and analyzed their relationship to the cloned Type II and III receptors using peptide antibodies. We used dithiothreitol and phosphoinositol-phospholipase C pretreatments to establish whether the 65 kDa labeled band which we observed corresponded to the Type I receptor or a glycophosphotidylinositol-linked binding protein. The results demonstrated that microvascular but not macrovascular endothelial cells express high levels of the Type III receptor. This differential expression of the Type III receptor indicates that distinct anatomical segments of the vasculature have distinct TGF-beta receptor profiles. The presence of the Type III receptor on micro- but not macrovascular endothelial cells may account for the reportedly different potency of TGF-beta 1 and TGF-beta 2 on these two endothelial cell types. Analysis of the 85 kDa and 65 kDa affinity-labeled bands revealed that all the endothelial cells express the Type II receptor and a band consistent with the presence of a dithiothreitol-sensitive Type I receptor. Two isoform-specific phosphoinositol-phospholipase C releasable TGF-beta binding proteins were also detected: a 60 kDa protein on one micro- (EJG) and one macro- (FBHE) vascular endothelial cell line and a 150/180 kDa protein on the macrovascular cell lines (FBHE and CPAE). These studies emphasize the heterogeneous nature of endothelial cells and underline the importance of using microvascular endothelial cells when examining TGF-beta responses related to microvascular function.
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PMID:Transforming growth factor-beta receptor expression on endothelial cells: heterogeneity of type III receptor expression. 755 2

A polyclonal rabbit antibody against 5'-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5'-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.
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PMID:Distribution of endogenous and exogenous 5'-nucleotidase on bovine spermatozoa. 792 8


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