Studies on the isolation and properties of renin granules from the rat kidney cortex.
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ABSTRACT:
SUBMITTER: Sagnella GA
PROVIDER: S-EPMC1161308 | biostudies-other | 1979 Aug
REPOSITORIES: biostudies-other
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Project description:1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.
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Project description:1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of alpha-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate alpha-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal alpha-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6.2. 4. The stability of lysosomal suspensions was studied. Incubation at 37 degrees and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the alpha-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6-7 and less stable at pH 8-9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0.6m to 0.25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.
| S-EPMC1206800 | biostudies-other
Project description:1. On isoelectric focusing, renin from rat kidneys showed three activity peaks with pI values at pH 5.0, 5.2 and 5.4 after a purification procedure involving differential centrifugation, acidification, chromatography on Sephadex G-75 and dialysis. 2. The preparation (purified 140-fold) was compared with a crude kidney extract in the absence and presence of 3 M-urea by isoelectric focusing. The pattern of activity distribution was confirmed by these experiments and the content of isoenzymes in the three groups calculated. 3. Pig renin was prepared and compared with rat renin with regard to molecular weight, acid activation, behaviour on isoelectric focusing, immunogenicity and substrate affinity. 4. Extracts of rat kidney contained multiple forms of renin with mol.wt. between 39000and 42000, whereas active pig renin had an approximate mol.wt. of 40000. Acidification of rat renal extracts did not increase the activity of renin, indicating the absence of an inactive form of renin in rat kidneys, whereas pig renin was activated by this procedure. Pig renin has isoelectric points at pH 4.6, 4.8, 5.05 and 5.2, significantly lower than for rat renin. The isoenzymes from the two species had no antigenicity in common, as shown by crossed immunoelectrophoresis or rocket immunoelectrophoresis. 5. The Michaelis constants for pig and rat renin were in the same range, 1 X 10(-6) M, when rat renin substrate was used. The relative content of rat isoenzyme with pI in the pH ranges 4.9-5.1, 5.1-5.3 and 5.3-5.5 was approx. 20, 27 and 53% respectively. Purified pig renin prepared in two different ways had isoenzymes with pI in the pH regions 4.5-4.7, 4.7-4.9, 4.9-5.05 and 5.05-5.20 in the approximate proportions 14, 24, 28 and 29%.
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Project description:The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.
| S-EPMC1164204 | biostudies-other
Project description:Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [(32)P]P(i)in vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [(32)P]P(i)in vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.
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Project description:1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.
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Project description:Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.
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Project description:When rat kidney cortex slices were incubated with glycine or [1-14C]glycine, after correcting for metabolite changes with control slices, product formation and glycine utilization fitted the requirements of the equation: 2 Glycine leads to ammonia + CO2 + serine. Evidence is presented that degradation via glyoxylate, by oxidation or transamination, is unlikely to have any significant role in kidney glycine catabolism. It is concluded that glycine metabolism in rat kidney is largely via glycine cleavage closely coupled with serine formation. 1-C decarboxylation and urea formation with glycine in rat hepatocyte suspensions were somewhat greater than decarboxylation or ammonia formation in kidney slices, showing that in the rat, potentially, the liver is quantitatively the more important organ in glycine catabolism. There was no evidence of ammonia formation from glycine with rat brain cortex, heart, spleen or diaphragm and 1-C decarboxylation was very weak.
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Project description:Pericytes, perivascular cells embedded in the microvascular wall, are crucial for vascular homeostasis. These cells also play diverse roles in tissue development and regeneration as multi-lineage progenitors, immunomodulatory cells and as sources of trophic factors. Here, we establish that pericytes are renin producing cells in the human kidney. Renin was localized by immunohistochemistry in CD146 and NG2 expressing pericytes, surrounding juxtaglomerular and afferent arterioles. Similar to pericytes from other organs, CD146+CD34-CD45-CD56- renal fetal pericytes, sorted by flow cytometry, exhibited tri-lineage mesodermal differentiation potential in vitro. Additionally, renin expression was triggered in cultured kidney pericytes by cyclic AMP as confirmed by immuno-electron microscopy, and secretion of enzymatically functional renin, capable of generating angiotensin I. Pericytes derived from second trimester human placenta also expressed renin in an inducible fashion although the renin activity was much lower than in renal pericytes. Thus, our results confirm and extend the recently discovered developmental plasticity of microvascular pericytes, and may open new perspectives to the therapeutic regulation of the renin-angiotensin system.
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Project description:Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes, and cancer. There is growing recognition of the importance of mitochondria both as targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semiautomated methods to disrupt tissue using kidney and muscle mitochondria preparations as exemplars. These methods use the Barocycler, the PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of overhomogenization. Mitochondria isolated using the PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of the PCT Shredder and the Barocycler to be used to purify mitochondrial preparations.
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