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A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone. 相似文献
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At 37 degrees C, human plasma kallikrein I follows Michaelis-Menten behaviour and exhibits a normal linear relationship between the initial velocity of hydrolysis of Ac-Pro-Phe-Arg-OMe,HCl and enzyme concentration in the range 0--150 pM. At temperatures of 30 degrees C and below substantial deviations from linearity are observed over the same enzyme concentration range. The temperature-dependent autoinhibition of kallikrein I activity is reversible and is not due to low-molecular-weight endogenous inhibitors or cofactors. The kinetic effect is apparently due to aggregation and can be abolished by the addition of sodium deoxycholate. 相似文献
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Radioactive proteins from Escherichia coli cell envelope fraction were separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis was carried out under several sets of conditions, and autoradiographs were obtained. Many of the proteins were separated at well-defined positions with good reproducibility. Some of the proteins moved relative to these stationary proteins depending at least two factors, i.e. the amount of proteins applied in the first dimension and the electric current applied in the second dimension. Among more than 200 spots, methyl-accepting chemotaxis protein and flagellin were identified by using labelled or cold preparations of these proteins as markers. Some of the spots were assigned to proteins from the outer membrane of the bacteria. The results provide a good foundation for comparative studies of membrane proteins from genetically altered strains of the bacteria. 相似文献
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Kallikreins compose a multigene family coding for a subgroup of serine proteases, which are involved in the processing of bioactive peptides. Two rat kallikrein-related genes, RSKG-7 (rat submandibular gland kallikrein gene 7) and RSKG-3, have been cloned and their sequences analyzed. RSKG-7 is approximately 4200 bases in length and consists of five exons and four introns. The 5' end region contains the variant CATAT box and TTTAAA box; the 3' end region contains the polyadenylation signal AATAAA. This gene encodes a putative 28,935-dalton preproenzyme of 261 amino acids (aa). The active enzyme consists of 237 aa and is preceded by a deduced signal peptide of 18 aa and a profragment of 6 aa. RSKG-3 is highly homologous to RSKG-7 in terms of its sequence and structure; it encodes a 28,730-dalton prepropeptide consisting of a signal peptide of 18 aa, a profragment of 6 aa, and an active peptide of 235 aa. Sequence comparisons of RSKG-7, RSKG-3, and other kallikrein-related enzymes reveal the key amino acid residues needed for both serine protease activity (His/Asp/Ser) and kallikrein-like cleavage specificity at basic amino acids. Northern blot analyses using specific oligonucleotide probes demonstrate that, among the 12 tissues studied, RSKG-7 and RSKG-3 are expressed in the rat kidney and submandibular gland. Castration of male rats results in a decrease in submandibular gland RSKG-7 mRNA, which can be restored to the normal level by treatment with thyroxine or testosterone. On the other hand, neither castration nor hormonal manipulation affects RSKG-7 mRNA levels in the kidney. 相似文献
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During the course of chromate-induced acute renal failure (ARF), urinary kallikrein excretion (UKE), a serine protease of distal tubule origin in the normal animal was decreased but tissue kallikrein concentration (TK) was increased, suggesting intracellular accumulation. Severe morphological lesions were observed in proximal tubular cells which showed brush border damage, numerous vesicles, necrosis and liquefaction of cytoplasmic material. Less marked changes were also present in distal tubules: large apical vacuoles and swollen mitochondria. Compared to normal rats, using the peroxidase-anti-peroxidase (PAP) method for light microscopy, greater kallikrein immunoreactivity was detected along the apical pole in distal tubules, on the membrane and in the cytoplasm as well as in the glomerulus. By immunoelectron microscopy, kallikrein was found in the connecting apical area, along the luminal, basolateral and basement membranes, in some vesicles, in Golgi apparatus and on ribosomes bound to endoplasmic reticulum. In the glomerulus, kallikrein was observed along the luminal surface of endothelial cell. After 14 days a progressive recovery of renal function, tissue morphology and UKE towards control values was observed. The presence of immunoreactive kallikrein in the glomerulus observed only during ARF confirmed the previous demonstration of kallikrein mRNA in the glomerulus. The cellular accumulation results more likely from a dysfunction of a general secretory mechanism due to cell membrane alteration than from a specific inhibition of kallikrein production and secretion. 相似文献
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Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from homogenized rat kidney cortex. Kallikrein was concentrated in the plasma-membrane fraction, but not in the brush border of the proximal tubules. Kininase II or angiotensin I-converting enzyme was localized in the brush-border membrane. It is suggested that kallikrein in the urine may originate from the plasma membrane of the distal tubules and the conversion of angiotensin I and the inactivation of bradykinin may occur on the lumen membrane of the proximal tubular cells. 相似文献
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Studies on rat sympathetic neurons developing in cell culture. I. Growth characteristics and electrophysiological properties 总被引:3,自引:0,他引:3
In this series of three papers, we describe electrophysiological and pharmacological studies on sympathetic principal neurons developing in cell culture. This paper is concerned with the methods for growing and recording from the neurons and with observations on some of their electrical properties. The succeeding papers are concerned with functional synapses which the neurons form with one another. Superior cervical ganglia of newborn rats were dissociated into single cells and small cell clusters, and the resulting cell suspension of principal neurons and a much smaller number of non-neuronal cells was cultured at low density in medium containing nerve growth factor (D. Bray, 1970, Proc. Nat. Acad. Sci. USA.65, 905–910;R. E. Mains and P. H. Patterson, 1973a, J. Cell Biol.59, 329–345). As in the previous studies the multiplication of the non-neuronal cells could be controlled so that the neurons grew in the presence of an increasing number of non-neuronal cells or in the virtual absence of other cell types. Another method for obtaining mixed cultures was to plate the initial cell suspension onto a preexisting layer of cells dissociated from some other tissue (e.g., heart). Neurons grown for 3 weeks or longer in the presence of non-neuronal cells had resting potentials, passive electrical properties, and action potentials generally similar to those reported for principal neurons of the superior cervical ganglia of adult rats. Through the use of tetrodotoxin, tetraethylammonium, and cobalt, evidence was obtained for the presence of potential-sensitive sodium, potassium, and calcium channels. Frequently the action potential was followed by a prolonged after-hyperpolarization whose properties suggested the presence of potassium channels controlled by calcium ions. When the neurons were grown in the absence of non-neuronal cells, the action potentials were similar, but the prolonged after-hyperpolarization was rarely seen, and the neurons usually discharged repetitively in response to a steady depolarization. 相似文献
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G Porcelli G B Marini-Bettòlo H R Croxatto M Di Iorio 《The Italian journal of biochemistry》1975,24(3-4):175-187
A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein. 相似文献
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The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1. 相似文献
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Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations. 相似文献
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The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18% by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per A280, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8 X 10(4) from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein. 相似文献
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J P Girolami C Orfila C Pecher G Cabos-Boutot J L Bascands J P Moatti A Adam A Colle 《Biological chemistry Hoppe-Seyler》1989,370(12):1305-1313
Acute renal failure (ARF) was induced in rat following a single injection of sodium chromate. A transient polyuria and a 10-fold decrease in glomerular filtration rate was immediately observed after sodium chromate administration. Urinary sodium and potassium excretion were reduced within 24 h and remained decreased for 8 to 10 days. Progressive recovery of normal renal functions, mainly electrolyte excretion and filtration rate was observed 12 days after sodium chromate administration. Urinary kallikrein excretion (UKE) was decreased only 48 h after sodium chromate administration. However the proportion of the active and inactive form excreted was unchanged. UKE remained also at a reduced level for 8 to 10 days and returned progressively to base-line level. The kallikrein content in the tissue was significantly increased immediately after sodium chromate administration and recovered normal values 12 days later. The increase of kallikrein in the tissue is more likely unspecific due to impaired protein transport than a specific stimulation of renal kallikrein biosynthesis. The decreased UKE may indicate a distal tubular reversible dysfunction in this ARF model. These reductions in electrolyte excretion, glomerular filtration and UKE were associated with selective morphological lesions. Whereas the glomeruli were intact, important damages affected proximal tubule cells which appeared necrotic and showed presence of vacuoles, liquefaction of cytoplasmic material and lost of microvilli. Less marked lesions were however observed in distal tubules, particularly large vacuoles were present at the apical poles of the tubule cells, the sites of kallikrein secretion. These distal damages may be involved in the increase of tissue concentration and in the decrease of UKE.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice 总被引:1,自引:0,他引:1
Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation. 相似文献
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