Kidney Membrane Fractions Research Articles

Angiotensin III: a potent inhibitor of enkephalin-degrading enzymes and an analgesic agent.

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: localization along the nephron and effect of corticosteroids.

To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO3-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO3-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO3-ATPase in kidney membrane fractions. HCO3-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO3-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO3-ATPase to vanadate indicates that it belongs to the F0-F1 class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO3-ATPase activity at the sites of urinary acidification.

Kininase one-an'-a-half: the newest member of the kininase family.

A kininase I-like enzyme (carboxypeptidase) was purified to homogeneity from human urine and compared to the 48,000 mol. wt. (48K) active subunit of carboxypeptidase N. The urinary carboxypeptidase had a mol. wt. of 73,000 in gel filtration and 76,000 in SDS-polyacrylamide gel electrophoresis. It had a pH optimum of 7.0 and differed from the 48K subunit in stability, susceptibility to trypsin, and enzymatic activity. The urinary enzyme did not cross-react with antibody to carboxypeptidase N in "Western blotting". Urine from a patient genetically deficient in plasma carboxypeptidase N (21% of normal) contained normal levels of urinary carboxypeptidase with similar properties to that from pooled human urine. Membrane fractions from several tissues contained a similar carboxypeptidase activity. The activity was highest in a microvillous membrane fraction from human placenta (65 nmol/min/mg with Bz-Gly-Lys as substrate). High specific activities were also found in membrane fractions of human kidney (18 nmol/min/mg) and lung (8 nmol/min/mg). The membrane-bound enzyme was distinguished from lysosomal and catheptic carboxypeptidases as well as "enkephalin convertase" by the use of specific inhibitors. These results show that urine contains a carboxypeptidase capable of cleaving arginine or lysine from the C-terminus of peptides. The enzyme does not arise from plasma carboxypeptidase N, but may be released into the urine from the renal brush border.

Displacement of 3H-EKC binding by opioids in rat kidney: A correlate to diuretic activity

Multiple opioid binding sites have been documented in brain tissue. In this study we report on the presence of binding sites for the opioid ethylketocyclazocine (EKC) in a membrane fraction of rat kidney. Binding appeared to be selective in that opioids varied markedly in their capacities to displace 3H-EKC. Correlating with the capacity of an opioid to displace 3H-EKC was the ability to produce diuresis. Although our studies cannot assign a particular physiological or pharmacological role for the renal EKC binding sites, binding studies of this nature may, nonetheless, be a means by which diuretic activity of opioids can be predicted.

Purification of a membrane-bound neutral endopeptidase from rat kidney and its activation by polyamines.

An endopeptidase which cleaves succinyl trialanine p-nitroanilide (Suc(Ala)3-pNA) into succinyl dialanine and alanine p-nitroanilide (Ala-pNA) was solubilized from a microsomal membrane fraction of rat kidney with Nonidet P-40 following treatment with 1 M KCl and Brij 35. The solubilized enzyme was purified to homogeneity by DEAE-Sephadex chromatography, Sepharose CL-6B gel filtration and sucrose gradient centrifugation. The final enzyme preparation had a specific activity of 1.69 mumol/min/mg protein, representing about 140-fold purification over the starting membrane. The enzyme hydrolyzes Suc(Ala)3-pNA with a Km value of 0.28 mM and a Vmax value of 1.3 mumol/min. The molecular weight of the undenatured enzyme was estimated to be 360,000 by gel filtration on a Sepharose CL-6B column and that of the denatured enzyme to be 92,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, revealing the presence of a single polypeptide chain. The enzyme was markedly activated by polyamines, producing increases in the values of both Km and Vmax. Comparatively less activation was found in the presence of some monovalent cations and Ca2+. The activation by polyamines was inversely proportional to the concentration of monovalent cations, but Ca2+ and polyamines seemed to stimulate additively.

Plasma antidiuretic hormone levels and kidney responsiveness to vasopressin in the jerboa, Jaculus orientalis

The plasma antidiuretic hormone (ADH) concentration and the kidney medulla responsiveness to vasopressin were measured in adult jerboas ( Jaculus orientalis) in different states of hydration. In 15 jerboas adapted to 30° and fed a dry diet, the average ADH concentration in blood plasma was 479 ± 59 pg/ml, as measured by a radioimmunoassay. About 6 hr after receiving a 5% body wt water load by gavage, the plasma ADH concentration fell to 130 ± 30 pg/ml in the 5 jerboas still producing hypertonic urine (1022 ± 267 mosmol/liter) and to 41.5 ± 8.4 pg/ml in the 6 jerboas producing hypoosmotic urine (157 ± 6 mosmol/liter). In vitro biochemical experiments were performed on the kidney medullas from two groups of 5 jerboas fed a dry diet (group I) or a water-enriched diet (group II), respectively, for 4 to 7 weeks. Compared to group II, group I animals exhibited (a) higher plasma ADH values, 372 ± 86 versus 76 ± 25 pg/ml; (b) higher urine osmolarities (3817 ± 638 versus 647 ± 90 mosmol/liter); (c) some decrease in [ 3H]lysine-vasopressin (LVP) binding capacity to kidney membrane fractions (maximal binding: 0.4 versus 0.6 pmol [ 3H]LVP bound/mg protein); d) decreased adenylate cyclase responses to arginine-vasopressin, lysine-vasopressin, and oxytocin in kidney membrane fractions; and (e) weaker adenylate cyclase responses to arginine-vasopressin in microdissected pieces of the medullary thick ascending limb of Henle's loop. The values found for (a) the dissociation constant of [ 3H]lysine-vasopressin binding to membranes ( K D ); (b) adenylate cyclase sensitivity to the three neurohypophyseal hormones ( K A ); and (c) adenylate cyclase sensitivity to arginine-vasopressin ( K A ) in medullary collecting tubules and medullary thick ascending limbs are similar in the two groups of jerboas and roughly comparable to those previously reported for the rat kidney medulla. The reduced maximal adenylate cyclase responses to vasopressin in the jerboas fed a dry diet might indicate some physiological “down regulation” of the number of vasopressin-specific receptors in the kidney as a result of the huge ADH concentration present in blood plasma under these conditions. However, this desensitization is not sufficient to account for the production of hypoosmotic urine in spite of the relatively high ADH plasma levels which persisted after acute overhydration.

The effect of some structural changes in the molecule of vasopressin on the duration of antidiuretic action

The effect of two structural variations in the molecule of [8-D-arginine]deamino-vasopressin on the prolongation of the antidiuretic action of the analogues in non-anesthetised rats was studied. The replacement of glutamine in position 4 of the peptide chain with a suitable amino acid has prolonged the half-life of the antidiuretic effect. A more prolonged antidiuretic effect was obtained when the stabilization of the cyclic structure of [8-D-arginine]deamino vasopressin was performed by replacement of the disulfidic bridge by the monocarba bridge. The binding and activating constants (adenylate cyclase) in a membrane fraction of the rat kidney were compared with the factors influencing the metabolic stability of the molecule of the individual analogues.

Studies on the structure of gamma-glutamyltranspeptidase. III. Evidence that the amino terminus of the heavy subunit is the membrane binding segment.

γ-Glutamyltranspeptidase solubilized with Triton X-100 from the membrane fraction of rat kidney was previously shown to contain a hydrophobic domain that anchors the enzyme to the membrane, and which may be localized in the aminoterminal portion of the heavy subunit. The light subunit does not associate with the membrane [Tsuji et al . (1980) J. Biochem . 87, 1567–1571]. In the current work, the membrane binding portion of γ-glutamyltranspeptidase was obtained by treating Triton-solubilized enzyme with papain. This treatment resulted in solubilization of about 8.8% of the protein mass of the Triton-solubilized γ-glutamyltranspeptidase from the heavy subunit. The product split from the heavy subunit had a molecular weight of 6,000 judging from its amino acid composition. The residual enzyme showed no loss of catalytic activity but became hydrophilic. The fragment removed from γ-glutamyltranspeptidase by papain treatment was not particularly hydrophobic and contained carbohydrate. The partial amino-terminal sequences and carboxyl-terminal amino acids of the heavy and light subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 and papain were determined. The light subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 and papain contained the same sequence, Thr-Ala-(X)-Leu, as an amino-terminal portion, but that of the heavy subunit of the form solubilized with Triton X-100 contained the sequence Met-Lys-Asn-Arg-Phe-Leu-Val-Leu-Gly-Leu-Val-Ala-Val-Val-Leu-Val-Phe-Val-Ile-Ile-Gly- and the sequence of the hydrophobic domain resembled that of the signal peptide which contributes to directing newly synthesized secretory protein to associate with the membrane of the endoplasmic reticulum. The papain-solubilized form had a completely different amino-terminal sequence, Gly-Pro-Pro-Leu-. The carboxyl-terminal amino acids of the subunits of the two forms were determined by carboxypeptidase digestion; that of both light subunits was found to be phenylalanine, and that of both heavy subunit to be tyrosine. These results indicate that the amino-terminal region of the heavy subunit of γ-glutamyltranspeptidase is removed by proteolysis with papain and that it is composed af a hydrophobic domain and a hydrophilic domain. The hydrophobic domain contains the amino-terminus of the heavy subunit, includes about 20 hydropholic amino acids and contributes to anchorage of the enzyme to the membrane. The hydropholic domain following the hydrophobic domain probably contains carbohydrate.

Prekallikrein, kallikrein and renin in membrane fractions of rat kidney.

Two different plasma membrane enriched fractions were isolated from the homogenized rat kidney by differential centrifugation in dextran or sucrose. Marker enzymes and morphological studies indicated that one fraction (BLM) was enriched in membrane particles originating from the basolateral membrane of tubular cells, while the other, the PM fraction, contained membrane from the luminal side. Membrane-bound kallikrein and renin were found in both fractions. Kallikrein activity was enhanced by phospholipase A2, melittin and detergents. Renin activity was greatly increased after solubilization by the same agents. In addition to bound kallikrein and renin BLM contained a prekallikrein which was activated by trypsin or plasmin. BLM prekallikrein has a slower electrophoretic mobility and a higher molecular weight than urinary or glandular kallikrein. The basal membrane of tubular cells appears to contain all of the essential enzyme components of the kallikrein and renin systems. Kallikrein of the PM fraction is probably released into the urine, while prekallikrein and kallikrein from basal membrane may be the source of kallikrein in lymph and renal venous effluent. Membrane-bound renin could be a form of renin retained by the kidney.

A trisialosyl ganglioside GT3 of hog kidney. Structure and biosynthesis in vitro.

A trisialosyl ganglioside was isolated from hog kidney cortex and purified with a yield of 6.8 nmol/g tissue or 13 mol % of total gangliosides. The homogeneous ganglioside was subjected to graded neuraminidase treatment, mild acid hydrolysis, periodate oxidation-borohydride reduction, permethylation analysis and chromium trioxide oxidation. The results suggested that the structure of this ganglioside is NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer (GT3). On the addition of exogenous disialosyl ganglioside GD3 and radioactive CMP-NeuAc, a GT3 synthesis activity was demonstrated in a crude membrane fraction of hog kidney. The activity was greatest in the presence of detergents and had a pH optimum of 6.4. Apparent Km values for CMP-NeuAc and GD3 were about 0. 6 and 0.1 mM, respectively. The reaction produce was assigned as GT3 by chromatographic separation, carbohydrate analysis and periodate oxidation followed by borohydride reduction.

A distinct peptidyl dipeptidase that degrades enkephalin: Exceptionally high activity in rabbit kidney

Peptidyl dipeptidase activity distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane fractions of rabbit kidney and lung. The enzyme cleaved Leu-enkephalin at the Gly-Phe bond, releasing Tyr-Gly-Gly and Phe-Leu, and also acted on bradykinin releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this peptidyl dipeptidase did not act on angiotensin I, or on hippuryl His-Leu, nor was it inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a K m for the kidney enzyme of 80 μM with Leu-enkephalin as a substrate. Our findings indicate that more than one enzyme is present in membrane preparations of lung and kidney inactivating enkephalin, and suggest a role for these enzymes in the peripheral actions of opiate and related peptides.

Evidence for interference of 25(OH)vitamin D3 with phosphaturic action of glucagon.

The effect of 25(OH)vitamin D3 [25(OH)D3] on the phosphaturic action of glucagon was studied using clearance techniques in the following groups of rats: group 1, parathyroidectomized (PTX) glucagon-infused rats receiving intravenous 25(OH)D3; group 2, PTX 25(OH)D3-pretreated rats receiving intravenous glucagon; and group 3, the thyroparathyroidectomized glucagon-infused rats receiving intravenous 25(OH)D3. The effect of 25(OH)D3 on glucagon-induced increase of cAMP in kidney slices and glucagon-activated adenylate cyclase (AC) in kidney membrane fractions was studied in vitro. In group 1, 25(OH)D3 suppressed the glucagon-induced phosphaturia by reducing fractional excretion of phosphorus (CP/CIn) from 0.175 +/- 0.02 (mean +/- SE) to 0.112 +/- 0.12 (P less than 0.05); this was associated with a reduction of urinary cAMP from 1,830 +/- 230 to 660 +/- 120 pmol/min (P less than 0.01). In group 2, pretreatment with 25(OH)D3 reduced CP/CIn from 0.221 +/- 0.025 to 0.108 +/- 0.012 (P less than 0.005). In group 3, 25(OH)D3 reduced CP/CIn from 0.165 +/- 0.012 to 0.075 +/- 0.011 (P less than 0.005). In vitro, 25(OH)D3 blunted the glucagon-induced activation of the AC/cAMP system by reducing AC from 570 +/- 30 to 325 +/- 28 pmol cAMP.mg protein-1.h-1 (P less than 0.01) and the cAMP level from 11.2 +/- 0.9 to 8.5 +/- 0.7 pmol cAMP/g wet tissue (P less than 0.05). These results show that 25(OH)D3 blunts the phosphaturic action of glucagon and suggest that this response may be mediated through suppression of the AC/cAMP system.

Prolactin-binding sites in tilapia ( Sarotherodon mossambicus) kidney

Highly purified 125I-labeled tilapia ( Sarotherodon mossambicus) prolactin (PRL) binds to microsomal membrane fractions prepared from tilapia kidney but not liver or gill. Specificity studies revealed that up to 35% of the labeled tilapia PRL bound to the kidney membrane preparation could be displaced by 100 ng of unlabeled hormone. Ovine PRL displaced 125I-labeled tilapia PRL from the kidney PRL-binding site. However, 125I-labeled ovine PRL demonstrated negligible specific binding to microsomal membrane fractions of tilapia kidney, liver, gill, or muscle. Tilapia PRL did not inhibit the binding of 125I-labeled ovine PRL to lactating rat liver membranes. The specific binding of 125I-labeled tilapia PRL to tilapia kidney membranes was greater with freshwater-acclimated than with seawater-acclimated fish suggesting that the tilapia kidney PRL-binding site is subject to physiological regulation.

A radioreceptor assay for purified teleost growth hormone

Highly purified 125I-labelled tilapia (Sarotherodon mossambicus) growth hormone (GH) binds to membrane fractions prepared from tilapia liver. Specific (displaceable) binding occurred with the 600, 15,000, and 90,000g fractions with the greatest binding observed with the 90,000g microsomal membrane fraction. Binding was dependent on time and membrane concentration. Specificity studies showed that up to 60% of the 125I-labelled tilapia GH bound to the liver microsomal membrane fraction could be displaced by 500 ng of unlabelled hormone. Scatchard analysis of the binding of 125I-labelled tilapia GH revealed a single class of binding site with a binding capacity of 125 ± 7.7 fmol/mg protein and a binding affinity of 1.5 × 1010 ± 0.4 × 1010 (SEM) l/mol. GH preparations from several vertebrate classes and tilapia prolactin and ovine prolactin at high concentrations displayed competition for the tilapia GH-binding site.125I-Labelled tilapia GH demonstrated specific binding to liver microsomal membrane fractions of the teleosts Gillichthys mirabilis, Salmo gairdneri, and Oncorhynchus tschawytscha but not to those of an amphibian Necturus maculosus or a bird Coturnix japonica. Slight, but significant, specific binding was observed with the microsomal membrane fraction of tilapia kidney and gill and of rat liver. These data suggest that this tilapia GH radioreceptor assay may have an application in the detection of teleost GH-like activity.

Metabolism and action of the prostaglandin endoperoxide PGH 2 in rat kidney

Kidney membrane fractions metabolized [1- 14C]PGH 2 to TXB 2, PGE 2, PGF 2α, PGD 2, 6-keto PGF 1α, and HHT. TXA 2, as measured by TXB 2, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH 2 caused a labile inhibition of cortical PGE 2-stimulated adenylate cyclase. PGE 2, PGF 2α, and PGD 2 are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH 2 inhibition suggested that TXA 2 was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH 2 is inhibition of adenylate cyclase.

Age related changes in the lipids of the microsomal and the mitochondrial membranes of rat liver and kidney

The lipid contents of the microsomal and mitochondrial membrane fractions of liver and kidney were determined in 6 and 24 month old rats. A significant age related increase in the molar ratio of cholesterol/phospholipid was observed in all membrane fractions. A significant age related reduction of phospholipid was noted in the microsomal fractions of liver and kidney. The relative amount of phosphatidylethanolamine was found to decrease in all membrane fractions during aging. Membrane glyceride content, however, remained relatively constant with age. Significant increase in oleic acid was seen in the neutral lipid of both liver and kidney and in the polar lipid of kidney. Significant increase in docosahexaenoic acid and significant decrease in linoleic acid were seen in the polar lipid of the liver membrane fractions. Possible alterations in membrane physiochemical properties and in membrane function due to these age related lipid changes are discussed.

The action of a binary nonionic detergent on a kidney membrane fraction

The disruption of a kidney cortex microsomal membrane preparation by a binary, nonionic detergent, was followed by using as markers, the changes in total protein content, and (Na +, K +)-ATPase in a supernatant fraction. Both markers responded similarly to changes in pH, microsome concentration and detergent concentration, but responded differently for time-dependent studies. The (Na +, K +)-ATPase activity was increased 2.2-fold (76.1 μmoles P i/mg protein/h, 95% ouabain-sensitive) by a single detergent treatment and 3.5-fold (92% ouabain-sensitive) by a sequential detergent treatment. Changes in the critical micelle concentration (cmc) were observed for varying detergent and protein concentrations, which suggest interactions of monomeric detergent with the membrane. The peak of (Na +, K +)-ATPase activity occurred above the cmc which suggests the participation of micelles in releasing the enzyme from the membranes. Hill plots of the protein released as the detergent concentration was varied showed a change in the slope near the cmc indicating a four-fold increase in the binding of detergent to membranes as the detergent concentration is increased above the cmc. These results suggest that the disruption of membranes by detergent involves the binding of detergent monomers to the membrane followed by the formation of co-micelles of the detergent with segments of the membrane to complete the separation process.

RADIORECEPTOR ASSAY FOR INSULIN

A sensitive and simple radioreceptor assay (RRA) capable of detecting 10 muU/ml of serum insulin has been developed using the crude membrane fraction of guinea pig kidney and 125I-porcine insulin. Using this assay system, serum insulin during 100 g oral glucose tolerance tests in six males including one obese man was measured. The mean concentration of serum insulin increased from 19 muU/ml during fasting to maximum of 90 muU/ml after 60 min of glucose load and then gradually declined to 38 muU/ml by 180 min. The values obtained by the present RRA agreed well with immunoreactive insulin (IRI).

ステロイドミオバチーマウスの骨格筋形質膜分画におけるOuabain感受性(Na<SUP>+</SUP>+K<SUP>+</SUP>)-ATPase活性

In order to observe the membrane property in steroid myopathy, the ouabain-sensitive (Na++K+)-ATPase activities of skeletal muscle, kidney, and brain membrane fractions were studied with the mice administered dexamethasone intraperitoneally for 7.0 -8.5 weeks at a dosage of 5.5 mg/kg. The results obtained were as follows. 1) The body weight of mice treated with dexamethasome for 7.0-8.5 weeks decreased by approximately 5 g (ca. 20% of the control body wt.) against that of saline-treated mice. 2) The ouabain-sensitive (Na++K+)-ATPase activity of skeletal muscle sarcolemmal fraction from the steroid-treated mice was significantly higher than that from control by 30-50%. 3) With kidney and brain membrane fractions, no differences were observed between control and steroid-treated mice in the ouabain-sensitive (Na++K+)-ATPase activity. These results show that the membrane property of skeletal muscle is specifically influenced by the long-term administration of dexamethasone.