Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Semisynthetic analogues of insulin were prepared from derivatives of desoctapeptide-(B23-30)-insulin (DOI). A1, B1-(Boc)2-DOI (di-Boc-DOI) was converted to A1, B1-(Boc)2-DOI-B22-phenylhydrazide (di-Boc-DOI-NHNH-C6H5) by the trypsin-catalyzed addition of phenylhydrazine in aqueous organic solvents at pH 6.5 [Canova-Davis, E., & Carpenter, F. H. (1981) Biochemistry 20, 7053-7058]. Treatment of di-Boc-DOI-NHNH-C6H5 with BNPS-skatole produced the phenyldiimide. The latter was coupled with a variety of protected peptides that, after removal of protecting groups, yielded the following compounds whose biological activities were compared to that of insulin in binding, in stimulation of hexose transport (), and in the stimulation of lipogenesis [)), in terms of percent of insulin activity, all in the isolated epididymal fat cell: di-Boc-DOI 0.2, (0.1), [0.2]; di-Boc-DOI-NHNH-C6H5 0.5, (0.2), [0.5]; DOI 0.2, (0.2), [0.1]; DOI-(Gly)B23 0.2, (0.2), [0.1]; DOI-(Gly-Phe)B23-24 6.3, (6.3), [8.0]; DOI-(Gly-Phe-Phe)B23-25 17.0, (25.6), [24.7]; DOI-(Gly-Phe-Phe-Tyr)B23-26 59.0, (50.0), [69.0]. The semisynthetic derivatives represent a stepwise readdition of the aromatic residues near the C terminus of the B chain. A given analogue demonstrated comparable activity in all three biological assays. The results indicate that the stepwise addition of aromatic residues to the B-chain C terminus of DOI produces an increase in insulin-like activity. The biological activity of DOI-(Gly-Phe-Phe-Tyr)B23-26, the derivative in which the aromatic region has been completely reassembled, is the same order of magnitude as that of insulin.
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PMID:Preparation of semisynthetic insulin analogues from bis(tert-butyloxycarbonyl)-desoctapeptide-insulin phenylhydrazide: importance of the aromatic region B24-B26. 634 Jul 39

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains chi 1776 and HB101 bearing plasmid p147 were partially purified and assayed for biological activity by determining stimulation of [1-14C]glucose oxidation to 14CO2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO2 production. Larger amounts of fused protein were obtained by transformation of strain PR13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.
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PMID:Biological activity of rat proinsulin synthesized by Escherichia coli. 634 Nov 71

Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column permitted the isolation of a polypeptide (Mr = 94,000) containing the beta-ketoacyl reductase activity but no other partial activities normally associated with the synthetase. The specific activity of the beta-ketoacyl reductase increased 2 to 3 times in this fraction, an increase that is within the expected range based on relative molecular weight. The kinetic parameters of this fraction towards NADPH and N-acetyl-S-acetoacetyl cysteamine were essentially the same as the beta-ketoacyl reductase component of the intact synthetase. However, the purified fragment did not catalyze the reduction of acetoacetyl-S-CoA derivative, a substrate that is readily reduced by the intact synthetase. Fluorescence measurements with etheno-NADP+ indicate the binding of about 1 mol of NADP+/94,000 daltons, a value which is in agreement with the results obtained from fluorescence measurements with NADPH and the binding of a radiolabeled photoaffinity analog of NADP+. Phenylglyoxal inhibits the beta-ketoacyl reductase activity of either the intact synthetase or the isolated fragment, suggesting the involvement of an essential arginine at or near the active site. Another fragment (Mr 36,000) containing beta-ketoacyl reductase activity was isolated from the synthetase after kallikrein/subtilisin double digestion. Previous mapping studies had shown that this fragment lies adjacent to the COOH-terminal thioesterase domain and overlaps the tryptic Mr 94,000 peptide by approximately 21 daltons. This fragment, but not the Mr 94,000 fragment, was found to contain the phosphopantetheine prosthetic group, indicating that the acyl carrier protein moiety is located in the 15,000-dalton segment that separates the beta-ketoacyl reductase from the thioesterase domain.
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PMID:The architecture of the animal fatty acid synthetase. III. Isolation and characterization of beta-ketoacyl reductase. 636 Oct 31

We examined the effects of elastase [EC 3.4.21.11] on lipogenesis, antilipolysis, and pyruvate dehydrogenase activity in rat epididymal adipose tissue in comparison with those of insulin and trypsin [EC 3.4.21.4]. The rate of conversion of [3-3H]-glucose into lipid in fat cells was stimulated by elastase, trypsin, and insulin. When fat pads were incubated with elastase, trypsin, or insulin in the presence of glucose, pyruvate dehydrogenase activity in the homogenate of the incubated fat pads was markedly increased. In the absence of glucose, elastase did not increase pyruvate dehydrogenase activity, though trypsin and insulin showed a slight but significant increase. Further, the increasing effect of elastase in the presence of glucose was inhibited by the addition of 3-O-methylglucose or phlorizin to the incubation mixture of the fat pads. Trypsin and insulin still showed a significant increase under similar conditions. When the homogenate of intact fat pads was incubated with elastase, the pyruvate dehydrogenase activity was progressively decreased with increase in the concentration of elastase. Concanavalin A showed an additive effect on the pyruvate dehydrogenase activity increase caused by elastase, whereas such an effect was not observed with insulin or H2O2. The stimulation of lipolysis by epinephrine in the fat cells was not suppressed by elastase, in contrast to trypsin and insulin. These results suggest that elastase reacts with the cell surface, facilitates glucose transport into the fat cells, and consequently affects glucose and lipid metabolism by somewhat different mechanisms from those of insulin and trypsin.
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PMID:Effect of elastase on glucose and lipid metabolism in rat fat cells. 639 99

Previous in vitro studies demonstrated that ATP-citrate lyase is phosphorylated by cyclic AMP-dependent protein kinase at peptide A, containing a phosphoserine residue, and by ATP-citrate lyase kinase at peptide B, containing both phosphoserine and phosphothreonine residues (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956). In the present study, trypsin-digested, radiolabeled ATP-citrate lyase from rat epididymal fat pads was analyzed by high performance liquid chromatography. Phosphorylation occurred at three amino acid residues within two different peptide sequences; one (peptide a) contained phosphoserine and the other (peptide b) contained phosphoserine and phosphothreonine. The retention times and molecular weights were the same for peptides a and A and peptides b and B. Isoproterenol action increased peptide a phosphorylation and, to a lesser extent, peptide b phosphorylation. Insulin action also increased peptide a phosphorylation, but did not increase peptide b phosphorylation.
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PMID:ATP-citrate lyase phosphorylation in rat adipose tissue. 663 Feb 12

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.
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PMID:Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma. 663 Feb 19

Approx. 90% of the highly motile goat cauda-epididymal spermatozoa lose motility completely due to removal of the surrounding epididymal plasma (EP) by washing with a modified Ringer's solution. Motility can however, be reconstituted in approx. 80% of these washed cells that lost motility during washing, with the addition of goat cauda-EP. The activity of the EP-factor(s) that cause reconstitution of motility in the washed immotile spermatozoa, is nondialysable and sensitive to the action of trypsin. The activity of the factor(s) is not dependent on exogenous Ca2+ (1 mM), Mg2+ (1.2 mM), cyclic AMP (5 mM) or dibutyryl cyclic AMP (5 mM). The novel system may thus serve as an excellent analytical tool to yield an insight into the molecular basis of the regulation of spermatozoal motility.
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PMID:Evidence for the reconstitution of motility by epididymal plasma-protein factor(s) in immotile washed spermatozoa from goat cauda epididymis. 665 Aug 87

Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable.
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PMID:On the mechanisms of inhibition of insulin action by small-molecular-weight trypsin inhibitors. 675 16

Anionic sites of rat epididymal spermatozoa were measured at pH 7.4 using tritiated polycationized ferritin. The spermatozoa from the caput region had 1.25 +/- 0.06 X 10(6) anionic sites per cell and a binding constant of 1.26 +/- 0.01 X 10(6) M-1. Spermatozoa from the cauda region had 1.50 +/- 0.09 X 10(6) anionic sites per cell and a binding constant of 4.84 +/- 0.82 X 10(6) M-1. The values were mean +/- s.d. The anionic sites were partly sensitive to treatments with neuraminidase, trypsin and Triton X-100.
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PMID:Measurement of anionic sites of rat epididymal spermatozoa using tritiated polycationized ferritin. 688 40

A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re-established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.
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PMID:A collagen-binding protein on the surface of ejaculated rabbit spermatozoa. 699 66


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