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1.
2.
Corpora lutea were collected from sheep on Days 6, 10, and 15 of the oestrous cycle and Day 25 of pregnancy and dissociated into single cell suspensions. Purified preparations of large and small luteal cells were prepared by elutriation on all days except Day 6. Basal progesterone production by large cells was 6-8-fold higher than by small cells (36-65 vs 6-9 fg/cell/min). Oxytocin secretion was maximal on Day 6 (1.0 fg/cell/min) and declined thereafter. The number of receptors for LH increased between Day 6 and Day 10 and the two cell types had an equal number of receptors on Days 10 and 15 (19,000-23,000). Large cells on Day 25 of pregnancy had fewer receptors (12,000) than did small cells (26,000). Progesterone secretion by small luteal cells from all days examined was stimulated by LH (0.01-1000 ng/ml) in a dose-dependent manner; maximum sensitivity to LH occurred on Day 10. Despite the presence of receptors for LH on large cells, LH failed to stimulate progesterone production. Basal production of progesterone by large and small cells, and the response of small cells to LH, was not influenced by day examined. Re-combinations of large and small cells from Day 10 synergized to increase progesterone secretion. Prostaglandin E-2 (0.1-1000 ng/ml) did not stimulate progesterone secretion by large or small cells. 相似文献
3.
Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes. 总被引:6,自引:3,他引:3
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Microvillar membranes derived from the brush border of the renal proximal tubule are very rich in peptidases. Pig kidney microvilli contain endopeptidase-24.11 associated with a battery of exopeptidases. The manner by which some neuropeptides are degraded by the combined attack of the peptidases of this membrane has been investigated. The contribution of individual peptidases was assessed by including inhibitors (phosphoramidon, captopril, amastatin and di-isopropyl fluorophosphate) with the membrane fraction when incubated with the peptides. Substance P, bradykinin and angiotensins I, II and III and insulin B-chain were rapidly hydrolysed by kidney microvilli. Oxytocin was hydrolysed much more slowly, but no products were detected from [Arg8]vasopressin or insulin under the conditions used for other peptides. The peptide bonds hydrolysed were identified and the contributions of the different peptidases were quantified. For each of the susceptible peptides, the main contribution came from endopeptidase-24.11 (inhibited by phosphoramidon). Peptidyl dipeptidase A (angiotensin-I-converting enzyme) was of less importance, even in respect of angiotensin I and bradykinin. When [2,3-Pro3,4-3H]bradykinin was also investigated at a lower concentration (20 nM), the conclusions in regard to the contributions of the two peptidases were unchanged. The possibility that endopeptidase-24.11 might attack within the six-residue disulphide-bridged rings of oxytocin and vasopressin was examined by dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ation and by reduction and carboxymethylation of the products after incubation. Additional peptides were only observed after prolonged incubation, consistent with hydrolysis at the Tyr2-Ile3 and Tyr2-Phe3 bonds respectively. These results show that a range of neuropeptides are efficiently degraded by microvillar membranes and that endopeptidase-24.11 plays a key role in this process. 相似文献
4.
Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid. 总被引:5,自引:2,他引:3
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A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed. 相似文献
5.
N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed. 相似文献
6.
7.
Proteins of the kidney microvillar membrane. The 130 kDa protein in pig kidney, recognized by monoclonal antibody GK5C1, is an ectoenzyme with aminopeptidase activity. 总被引:6,自引:3,他引:3
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The hybridoma GK5C1, secreting a monoclonal IgG1 antibody, was generated after immunizing a mouse with pig kidney microvillar membranes. An immunoradiometric assay showed that only kidney and intestine contained detectable amounts of the antigen recognized by the antibody, the highest concentration being observed in the ileum. Immunocytochemistry confirmed this observation and revealed that the antigen was associated with renal and intestinal brush borders. By 'Western' blotting, the antigen in kidney microvilli was shown to be a 130 kDa polypeptide. Papain treatment of the membrane before blotting converted the antigen to a 125 kDa polypeptide, no longer associated with membrane. Immunoaffinity chromatography of detergent-solubilized kidney membranes yielded a pure 130 kDa protein. When one purification was monitored by the immunoradiometric assay, the yield was 3.5% and the purification factor was 1000-fold. The antigen constituted about 0.8% of the microvillar membrane protein. The protein could be reconstituted into liposomes, where electron microscopy revealed an asymmetric orientation, similar to that of ectoenzymes in this membrane. The stalk length was about 3 nm. In electron micrographs the purified protein appeared to be dimeric. A search for enzymic activity was rewarded when L-leucyl-L-tryptophan was observed to be hydrolysed. Failure to hydrolyse N-blocked peptides and the ability to release the N-terminal residue from extended peptides, including Leu-Trp-Leu and Leu-Trp-Met-Arg, showed that the activity was that of an aminopeptidase. The enzyme was maximally active at pH 7.5 and irreversibly inactivated outside the range pH 6-10. This activity could not be attributed to trace contamination with aminopeptidase N. The best substrates so far identified for the 130 kDa protein were those with tryptophan in the P1', position. This protein is a new microvillar enzyme and it is proposed that it be called aminopeptidase W. 相似文献
8.
S H Clarke J J Kenny D G Sieckmann S Rudikoff 《Journal of immunology (Baltimore, Md. : 1950)》1984,132(3):1544-1549
Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments. 相似文献
9.
Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil. 相似文献
10.