Purification and partial characterization of a thiol proteinase activating prokallikrein from the rat kidney cortex

The rat kidney cortex contains at least three kinds of prokallikrein-activating proteinase, and among these the one with the highest molecular weight was purified by a procedure including chromatography on CM-cellulose, concanavalin A-Sepharose, organomercurial-Sepharose 4B and Sephadex G-100. The resulting preparation was apparently homogeneous, as assessed by SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated to be 57,000. The optimal pH for the activation of prokallikrein by the preparation, termed activator I, was around 4.5. Activator I was inhibited by E-64, iodoacetate and leupeptin, but not by PMSF and phosphoramidon. In immunodiffusion analysis, the antiserum to activator I formed an immunoprecipitin arc with the extract from the kidney cortex or submandibular gland, but not with that from the pancreas. These results indicate that activator I is a thiol proteinase with a molecular weight of 57,000. A proteinase immunologically identical with activator I appears to be present in the submandibular gland.     

Purification and characterization of two forms of cytochrome P-450 from rat kidney cortex microsomes

Two forms of cytochrome P-450 (P-450), designated P-450 k-1 and P-450 k-2, have been purified about 100-fold from rat kidney cortex microsomes. P-450 k-1 and P-450 k-2 have monomeric molecular weights of 51,500 and 52,000, respectively, on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Absolute spectra of the oxidized forms indicate that P-450 k-1 is largely in the low-spin state and partly in the high-spin state, and that P-450 k-2 is essentially all in the former. The absorption maxima in reduced carbon monoxide difference spectra are at 450.5 and 451 nm with P-450 k-1 and P-450 k-2, respectively. The two P-450s catalyze the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, although P-450 k-1 exhibits a higher specific activity with all fatty acids tested. In addition, P-450 k-1 is capable of hydroxylating prostaglandin (PG) A1 and A2 at the omega-position, whereas P-450 k-2 has no activity toward PGs. These activities are all stimulated by addition of cytochrome b5. The two P-450s give different peptide map patterns when partially digested with Staphylococcus aureus V8 protease or papain.     

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at -20 degrees C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The Km for the donor, 3'-phosphoadenosine 5'-phosphosulfate, is 5.2 microM. Structural studies on this "active" sulfate donor show the requirement of a phosphate group at the 3' position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.     

Purification and characterization of the activated mineralocorticoid receptor from rat kidney

1. The mineralcorticoid receptor (MR) from rat kidney was purified within 8 hr by the following, successive steps: stabilization with synthetic, tritiated steroids (RU 26752 or R 5020), phosphocellulose passage, heat activation (25 degrees C), and DNA-cellulose batch elution. 2. The purified preparation was resolved as a single, 75 KDa band on SDS-PAGE electrophoresis although the exact degree of purity was difficult to assess by the charcoal assay due to denaturation. 3. The natural hormone, aldosterone, was unsuitable for receptor purification and characterization. 4. The MR purified with different ligands behaved identically during ion exchange and gel permeation analyses, suggesting post-translational modifications of the native receptor in whole cytosol that exhibits molecular heterogeneity.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.     

Isolation and characterization of autophagic vacuoles from rat kidney cortex

The number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues. The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparent vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.     

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anion-exchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 microM and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [14C]palmitate binding to the protein.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity

"Enkephalinase," a membrane-bound peptidase hydrolyzing the Gly3-Phe4 amide bond of enkephalins, initially characterized in brain, was purified from a rat kidney microsomal fraction. After differential solubilization with Triton X-100, the use of DEAE-Sephadex, concanavalin A, and hydroxylapatite chromatography led to a 2000-fold purification, close to homogeneity. Renal enkephalinase appears to be a glycoprotein Mr = 92,000-95,000 with catalytic properties and sensitivity to chelating agents and inhibitors (Thiorphan, phosphoramidon) very similar to those of the cerebral enzyme. The enzyme co-purified until the final step with "renal brush-border neutral proteinase" (EC 3.4.24.11) activity assayed with 125I-insulin B chain as substrate and displaying similar sensitivity to inhibitors. The specificity of the purified enkephalinase has been studied using either peptides derived from the enkephalins or model peptides of general formula (Ala)m-Tyr-(Ala)n as substrates. In all cases the bond cleaved was that involving the amino group of an aromatic residue, specificity being also defined by the nature of the neighboring residue on the COOH-terminal side. A free carboxyl in the latter residue was essential in the two series of substrates, indicating that enkephalinase more efficiently functions as a dipeptidyl carboxypeptidase than as an endopeptidase.     

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).     

Kinetic characterization of phosphofructokinase isolated from rat kidney cortex.

1. Phosphofructokinase from rat kidney cortex has been purified by affinity chromatography to a final specific activity of 15 units per mg of protein, measured at 25 degrees C and pH 8. 2. This lower spec. act., compared with that of the enzyme from other sources, shows the enzyme in proximal tubules to be less active, which would account for the main gluconeogenic role of these nephron sections. 3. The binding of fructose-6-phosphate to the enzyme is co-operative. ATP increases the Hill coefficient and produces a marked allosteric inhibition on the activity. 4. Fructose-2,6-bis-phosphate is a potent activator of the enzyme from this source. It reduces the Hill coefficient of the enzyme and the inhibition constant of ATP. A marked difference between this and the liver enzyme is that the activation is not co-operative.     

Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex.

Glucosamine-6-phosphate deaminase (EC 5.3.1.10) from dog kidney cortex was purified to homogeneity, as judged by several criteria of purity. The purification procedure was based on two biospecific affinity chromatography steps, one of them using N-epsilon-amino-n-hexanoyl-D-glucosamine-6-phosphate agarose, an immobilized analog of the allosteric ligand, and the other by binding the enzyme to phosphocellulose followed by substrate elution, which behaved as an active-site affinity chromatography. The enzyme is an hexameric protein of about 180 kDa composed of subunits of 30.4 kDa; its isoelectric point was 5.7. The sedimentation coefficient was 8.3S, and its frictional ratio was 1.28, indicating that dog deaminase is a globular protein. The enzyme displays positive homotropic cooperativity toward D-glucosamine-6-phosphate (Hill coefficient = 2.1, pH 8.8). Cooperativity was completely abolished by saturating concentrations of GlcNAc6P; this allosteric modulator activated the reaction with a typical K-effect. Under hyperbolic kinetics, a Km value of 0.25 +/- 0.02 mM for D-glucosamine-6-phosphate was obtained. Assuming six catalytic sites per molecule, kcat is 42 s-1. Substrate-velocity data were fitted to the Monod's allosteric model for the exclusive-binding case for both substrate and activator, with two interacting substrate sites. The Kdis for N-acetyl-D-glucosamine-6-phosphate was estimated at 14 microM.     

Hydrodynamic and biochemical correlates of the activation of the glucocorticoid-receptor complex

Possible changes in the size and shape of the glucocorticoid-receptor complex (GRC) following activation remain poorly documented, due to the lability and possible activation of the receptor during the determination of these hydrodynamic parameters. In the present study molybdate was used to stabilize the GRC, thus preventing these uncontrolled transformations. Cytosol prepared from mouse whole brains was incubated for 18 h at 0-2 degrees C with [3H]triamcinolone acetonide (+/- molybdate). Activation was then initiated by incubation at 22 degrees C for variable times and quenched at 0 degree C by adding molybdate. The Stokes radius and sedimentation coefficient of the GRC declined from 77 A and 9.2 S before activation to 58 A and 3.8 S after activation. These measurements remained consistent after recycling GRC between sedimentation and gel filtration procedures and correspond to a 3-fold reduction in the relative molecular mass. The loss and formation of the 297 and 92 kDa species, respectively, after different durations of activation correlated nearly perfectly with increased binding of GRC to DNA-cellulose (DNA-C). The observed size change also correlated well with decreased adsorption to DEAE-cellulose filters (DE-81) and increased adsorption to glass fiber filters (GF/C). The increased adsorption to GF/C may reflect an increase in hydrophobicity which, with extended durations of activation, leads to increased aggregation and reduced binding to DNA-C, but not to a change in adsorption to DE-81. We propose that during activation the 297 kDa form of the GRC splits to form a 92 kDa species that displays an increased affinity for DNA.     

Isolation and characterization of beta-glucosidase from the cytosol of rat kidney cortex.

A procedure is described for the preparation of extensively purified beta-D-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the beta-glucosidase in the high speed supernatant (100 000 X g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. beta-Glucosidase activity co-chromatographs with beta-D-galactosidase, beta-D-fucosidase, alpha-L-arabinosidase and beta-D-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of beta-glucosidase, respectively. The specific activity of the apparently homogeneous beta-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-beta-D-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6--7) and heat lability, and co-migrate on polyacrylamide disc gels at pH 8.9 (RF, 0.67). beta-Glucosidase acitivity is inhibited competitively by glucono-(1 leads to 5)-lactone (KI, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of beta-D-glucose, it will not hydrolyze xylosyl-O-serine, beta-D-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000--58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of beta-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convoluted tubule. The enzyme is also present in relatively large amounts in the villus cells, but not crypt cells, of the intestine. The physiological substrate and function of the enzyme are unknown.     

Purification and state of activation of rat kidney phenylalanine hydroxylase

Phenylalanine hydroxylase, the enzyme that catalyzes the irreversible hydroxylation of phenylalanine to tyrosine, was purified from rat kidney with the use of phenyl-Sepharose, DEAE-Sephacel, and gel permeation high pressure liquid chromatography. Our most highly purified fractions had a specific activity in the presence of 6-methyltetrahydropterin, of 1.5 mumol of tyrosine formed/min/mg of protein, which is higher than has been reported hitherto. For the rat kidney enzyme, the ratio of specific activity in the presence of 6-methyltetrahydropterin to the specific activity in the presence of tetrahydrobiopterin (BH4) is 5. By contrast, this ratio for the unactivated rat liver hydroxylase is 80. These results indicate that the kidney enzyme is in a highly activated state. The rat kidney hydroxylase could not be further activated by any of the methods that stimulate the BH4-dependent activity of the rat liver enzyme. In addition, the kidney enzyme binds to phenyl-Sepharose without prior activation with phenylalanine. The phenylalanine saturation pattern with BH4 as a cofactor is hyperbolic with substrate inhibition at greater than 0.5 mM phenylalanine, a pattern that is characteristic of the activated liver hydroxylase. The molecular weight of the rat kidney enzyme as determined by gel permeation chromatography is 110,000, suggesting that the enzyme might be an activated dimer. We conclude, therefore, that phenylalanine hydroxylases from rat kidney and liver are in different states of activation and may be regulated in different ways.     

Activation of the glucocorticoid-receptor complex

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.     

Effects of sodium tungstate on the nuclear uptake of glucocorticoid-receptor complex from rat liver

Effects of sodium tungstate on the nuclear uptake of rat liver cytosolic glucocorticoidreceptor complex were examined at pH 7. The nuclear uptake of heat-activated [³H]triamcinolone acetonide-receptor complex was blocked completely in the presence of 1 mm tungstate. A preincubation of nuclear preparation with tungstate (>0.1 mm) blocked the subsequent uptake of [³H]triamcinolone acetonide-receptor complex. When the tungstate-treated nuclear preparation was washed with 0.3 M KCl, its [³H]triamcinolone acetonide-receptor complex binding capacity recovered to 50% of that of control samples with no tungstate treatment. A preincubation of chromatin with tungstate yielded similar results. The nuclear-bound [³H]triamcinolone acetonide-receptor complex, formed either by an in vivo administration of [³H]triamcinolone acetonide or by an in vitro incubation of glucocorticoid-receptor complex with isolated nuclei, was extracted by tungstate in a concentration-dependent manner. The majority of nuclear-bound [³H]triamcinolone acetonide could be extracted with 0.1 and 1 mm tungstate from in vitro- and in vivo-labeled nuclei, respectively. The tungstate-extracted steroid-receptor complexes sedimented in 4–5 S and 3.3–3.5 S region in 10 mm KCl- and 0.3 mm KCl-containing sucrose gradients, respectively. Tungstate treatment caused an irreversible loss of the nuclear binding capacity of [³H]triamcinolone acetonide-receptor complex which could not be recovered after dialysis. These studies indicate that tungstate affects both glucocorticoidreceptor complex and certain nuclear or chromatin proteins.     

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.