A simple method of isolating tonin using chromatofocusing.
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Animals↗
Biomedical subjects
B J Morris
Publications and source records attributed to B J Morris.
Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases.
A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.
Amino Acid Sequence↗
The effect of bromocriptine on circulating vasopressin.
1. The oral administration of 2.5 mg of the dopamine (3,4-dihydroxyphenethylamine) agonist bromoergocriptine enhanced the osmotically stimulated rise in plasma [arginine]vasopressin ([Arg]VP) concentrations in five normal human subjects. 2. This finding lends support to the suggestion that the osmotically induced release of [Arg]VP is under dopaminergic control in man.
Adult↗
Clearance and metabolism of 125I-labeled tonin in the rat.
125I-Labeled rat tonin was injected iv into male Wistar rats weighing 303 +/- 17 g (mean +/- SE), and the disappearance from the circulation, plasma interaction, tissue uptake, and metabolism of radioactivity was studied. After injection, 125I-labeled tonin was immediately bound by alpha 1-macroglobulin, the major circulating protease inhibitor of rats, and the complex was cleared in a biexponential manner: t1/2 of the fast component = 1.8 +2- 0.1 min; t1/2 of the slow component = 84 +/- 10 min; MCR = 2.1 +/- 0.2 ml/min (mean +/- SE, n = 8). The complex was taken up intact by tissues, the major ones being the liver, spleen, adrenal, thyroid, and kidney. Analysis of tissue extracts by gel filtration on Sephadex G-100 showed that the kidney was the major site of metabolism, and radioactivity similar in size to 125I appeared rapidly in urine. Therefore tonin circulates wholly as a complex with alpha 1-macroglobulin (the rat equivalent of human alpha 2-macroglobulin), which once formed is quickly eliminated from the bloodstream by tissue uptake and metabolized.
Adrenal Glands↗
Biosynthesis of preprorenin. Studies using whole tissue, a cell-free system, and E. coli containing cDNA inserted at the PstI site of plasmid pBR322.
The biosynthesis of renin as a higher molecular weight 'prorenin' was demonstrated by in vitro incorporation of [35S] methionine into nascent polypeptides of submandibular gland tissue from adult male mice. Immunoprecipitation with anti-renin and electrophoresis identified a Mr 44,500, pI 6.4 prorenin which normally represented 5% of renin-immunoreactive protein in tissue extracts and which was rapidly converted during in vitro labeling into a Mr 40,000, pI 6.2 species. The latter was subsequently processed slowly to forms of Mr 35,500, pI 5.6 and Mr 34,000, pI 5.4. The influence of processing enzymes was then eliminated by synthesizing renin in a cell-free translation system containing rabbit reticulocyte lysate and mRNA isolated from submandibular glands. This yielded an even larger species of Mr 46,000 likely to be 'preprorenin'. A clone bank of mouse submandibular gland cDNA was prepared. This consisted of E. coli RRI transformed with plasmid pBR322 in which cDNA had been inserted at the PstI site so that the bacteria could express the encoded polypeptide. Renin-immunoreactive colonies were identified suggesting the expression of renin-like proteins by a prokaryote. The cDNA which was 700-1000 base pairs big was excised for sequencing.
Animals↗
Renin synthesis and gene cloning.
1. The present study describes the biosynthesis and gene cloning of mouse submandibular gland renin. 2. After translation of mRNA coding for renin a Mr 46 000 protein is produced (preprorenin). However, rapid removal of the 'pre' segment from the nascent chain would account for the fact that in pulse-chase and continuous labelling experiments the largest renin-immunoreactive protein seen was of Mr 44 500, pI 6.4 (determined by 2-dimensional gel electrophoresis). This 'prorenin' was rapidly converted to a Mr 40 000, pI 6.2 species and then more slowly to forms of Mr 35 500, pI 5.6 and Mr 34 000, pI 5.4, which correspond in Mr and pI to renin isolated in pure form from mouse submandibular glands. 3. Colonies of the bacterium Escherichia coli strain RRI, containing plasmid pBR322 into which mouse submandibular gland cDNA had been inserted at the PstI site, were screened with affinity-purified anti-renin. Of 500 colonies, two were found that reacted positively with the antibody. These contained protein that could be immunoprecipitated with anti-renin and cDNA which was capable of inter-colony hybridization and which had a HinfI restriction site in each case.
Animals↗
Evidence that the arginine esteropeptidase (gamma) subunit of nerve growth factor can activate inactive renin.
The beta and gamma subunits of nerve growth factor were isolated and tested for inactive-renin-activating activity using human amniotic fluid as a convenient source of inactive renin. Only the gamma subunit, which is an arginine esteropeptidase, could activate inactive renin. The role of this enzyme in sites containing both it and inactive renin warrants investigation.
Amniotic Fluid↗
Mechanism of activation of inactive renin in human plasma by puff adder venom.
Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.
Animals↗
Kallikrein and renin: molecular biology and biosynthesis.
1. A cDNA clone library was constructed from male mouse submandibular gland poly(A)+RNA. 2. A 500 base-pair sequence consisting of a 3' untranslated region plus a 447 base-pair region coding for an amino acid sequence having 57% homology with the C-terminal 149 amino acids in the 230 amino acid chain of porcine pancreatic kallikrein was identified. 3. The sequence was strongly but not completely homologous with known mouse submandibular gland serine proteinase sequences and represents the first report of a DNA base sequence for a serine proteinase. It may be part of the gene coding for kallikrein. 4. The biosynthetic pathway for renin was established by continuous-labelling, pulse-chase and cell-free translation studies of submandibular gland tissue from normal and testosterone-induced mice. 5. Renin was synthesized as a mol. wt. 46000 preprorenin which is likely to be hydrolysed before completion of the nascent chain. A prorenin of mol. wt. 44500, pI 6.4 was identified and shown to be rapidly converted into a mol. wt. 40000, pI 6.2 renin, which was then converted more slowly into forms of mol. wt 35500, pI 5.6 and mol. wt. 34000, pI 5.4.
Amino Acid Sequence↗
Renin in mouse but not in rat submandibular glands.
Renin was found in the submandibular glands of male Quackenbush mice in concentrations higher than has been reported for any tissue of any strain or species. However, no renin-like activity could e detected in glands from male and female Wistar rats using either pH 5.8 or 7.4 for assay and a radioimmunoassay specific for renin's reaction product, angiotensin I. Rabbit submandibular glands contained renin.
Animals↗
Activation of inactive renin during the selective destruction of proteinase inhibitors in human plasma by a metalloproteinase in Bitis arietans venom.
Puff adder venom, which has been pretreated with phenylmethylsulphonyl fluoride and extensively dialysed, is capable of destroying selectively proteinase inhibitory activity in human plasma by an action of an EDTA-sensitive venom proteinase. We found that incubation of 1/5 vol. of such venom with human plasma at 25 degrees C leads to a concomitant increase in renin to 4.4 times control by 5 h. The activation of inactive renin was abolished by 10 mM EDTA and the rate of activation was reduced by 50% in the presence of 5 mM phenylmethylsulphonyl fluoride and by 90% when 0.32 mg/ml soybean trypsin inhibitor and 5 mM N-ethylmaleimide were added as well. The venom proteinase thus appears to activate inactive renin via an activation of endogenous plasma proteinases. This may be accomplished either by activation of proteinase precursors or action on proteinase inhibitor-proteinase complexes. By destroying proteinase inhibitors at the same time as it activates endogenous proteinases, Bitis arietans metalloproteinase would appear to be particularly useful for studies of the activation of inactive renin in human plasma, since endogenous proteinases are then free to activate inactive renin without subsequent inhibition by endogenous proteinase inhibitors.
Animals↗
Effect of angiotensin II and sodium depletion on angiotensinogen production.
An in vitro preparation of liver slices was used to study the effect of angiotensin II and sodium depletion on the synthesis of angiotensinogen in rats. Two other treatments known to increase plasma angiotensinogen concentration in vivo, viz., intraperitoneal administration of dexamethasone or ethinyl estradiol, resulted in an increase in the rate of release of angiotensinogen by liver slices; this increase was inhibited by adding actinomycin D or vincristine to the incubation medium. Intravenous infusion of angiotensin II (33 ng/min for 3 days) also produced a marked increase in the release of angiotensinogen concentration and a decrease in plasma renin activity. In contrast, no change in the rate of release of angiotensinogen was observed in rats depleted of sodium for 7--14 days, even though these animals exhibited a marked increase in plasma angiotensin II concentration. Plasma angiotensinogen concentration decreased by 30%, presumably as a consequence of the accompanying increase in renin secretion. These results provide further evidence that the synthesis of angiotensinogen may be increased by angiotensin II, but indicate that the circulating level of angiotensin II in sodium-deficient animals is not sufficiently high to produce this response.
Angiotensin II↗
Inhibition by alpha-adrenoceptor agonists of renin release in vitro.
A variety of alpha-adrenergic agonists encompassing a broad range of concentrations were used to investigate the existence and nature of a putative alpha-adrenergic mechanism inhibitory to renin release, which may operate at the level of the juxtaglomerular apparatus. For this purpose rat renal cortical tissue incubated in vitro was used. Concentrations of noradrenaline, adrenaline and methoxamine of 10(-6), 10(-5), 10(-4) and 10(-3) M caused significant dose-related inhibition of renin release. The inhibition of release by these doses was reversed completely by 10(-4) M phentolamine. In contrast, phenylephrine, oxymetazoline and clonidine did not inhibit renin release. The results support the concept of an alpha-adrenergic mechanism inhibitory to renin release and show that high concentrations of alpha-adrenergic agonist are required for its operation in vitro. The manner in which this inhibitory mechanism affects renin release under physiological circumstances remains to be demonstrated.
Adrenergic alpha-Agonists↗
Partial purification of dog angiotensinogen.
Dog angiotensinogen was purified 450-fold from the plasma of nephrectomized dogs by a simple four-step procedure involving precipitation between 1.5 and 2.3 M ammonium sulfate, gel filtration on Sephadex G-150, ion-exchange chromatography on DE-52 cellulose, and affinity chromatography on Concanavalin A-Sepharose. The purity of the final preparation was over 50%. The preparation of dog angiotensinogen had an apparent molecular weight of 80,000 determined by gel filtration on Sephadex G-100. Kinetic studies indicated that the Km of the reaction of dog renin with partially purified dog angiotensinogen (1,840 pmol/ml) was similar to that for the reaction with angiotensinogen in diluted dog plasma (1,820 pmol/ml). Thus the purification procedures employed did not alter the affinity of dog renin for the Leu10-Leu11 bond of dog angiotensinogen. Because the concentration of angiotensinogen in dog plasma is about 700 pmol/ml, a first order reaction with respect to substrate is indicated in vivo.
Angiotensinogen↗
Localization of angiotensinogen in rat liver by immunocytochemistry.
Plasm angiotensinogen, the protein precursor of angiotensin, is produced by the liver. The present study investigated the location of angiotensinogen in sections of rat liver using the unlabeled peroxidase-antiperoxidase bridge technique of Sternberger. Specific reaction products of the antibody localization method were most pronounced in the cytoplasm of hepatocytes in the pericentral zone of the liver lobule. Controls, in which antibody was preabsorbed with angiotensinogen, did not form reaction product. The gradient of angiotensinogen observed within liver lobules resembled that demonstrated by metabolic zonation. The distribution of angiotensinogen differed from the scattered distributions found for other plasma proteins.
Angiotensinogen↗
Properties of the activation by pepsin of inactive renin in human amniotic fluid.
1. The renin present in human amniotic fluid was found to have an apparent Mr of 58 000 by gel filtration and is thus bigger than renin in untreated kidney extracts and plasma (Mr approximately 40 000). 2. Treatment with pepsin (40 microgram/ml pH 4.8, 2 h, 22 degrees C) caused a 6-fold increase in activity of this renin species, although Mr was not very different (57 000). 3. Unlike renal renin, renin in human amniotic fluid was not a glycoprotein and behaved similarly on concanavalin A-Sepharose before and after activation by pepsin. 4. Ion-exchange chromatography demonstrated a small change in the ionization properties of human amniotic fluid renin after activation by pepsin. 5. Pepsin-mediated activation resulted in a five-fold increase in V, but only a small decrease in the Km of renin to 39% of normal, so that the increase in activity observed was not due to an increase in the affinity of the enzyme for its substrate. The kinetic data were consistent with the theory of noncompetitive inhibition. 6. The activation of human amniotic fluid renin by pepsin may be caused by a change in the tertiary structure of the molecule subsequent to a proteolytic action that does not remove detectable polypeptide components.
Amniotic Fluid↗
The renin-angiotensin system.
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Adrenocorticotropic Hormone↗
The distribution of angiotensinogen in dog brain studied by cell fractionation.
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Acid Phosphatase↗