Stabilization of mammalian liver branched-chain α-ketoacid dehydrogenase by thiamin pyrophosphate

Thiamin pyrophosphate, CoASH, and NAD⁺ have been shown to reversibly bind to the purified bovine liver mitochondrial branched-chain α-ketoacid dehydrogenase complex. When saturated with thiamin pyrophosphate, the complex was more stable to heat and chymotrypsin inactivation. Under identical saturating conditions a conformational change in the complex was observed by circular dichroism spectroscopy. We postulate that thiamin pyrophosphate can increase the biological half-life of the in vivo, membrane-bound complex through conformational changes induced by the binding of this cofactor.     

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADH_SS. The ω-hydroxyfatty acid dehydrogenase activity of LADH_SS was of similar magnitude as that of LADH_EE whereas the ω-hydroxysteroid dehydrogenase activity of LADH_SS was considerably higher than that of LADH_EE.     

Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

Studies on the oxidation–reduction systems of the erythrocyte

1. Starvation for 3 days produces a decrease in methaemoglobin-reductase and glutathione-reductase activities, but it does not alter the glucose 6-phosphate-dehydrogenase activity of the rat erythrocyte. 2. The feeding of a protein-free diet for 11 days causes greater changes in the first two enzymes and also a diminution of the third. Under this experimental condition slight decreases in protein and haemoglobin contents were noted. 3. The experimental animals did not show methaemoglobinaemia, probably because the activity of methaemoglobin diaphorase is preserved. 4. The GSH content was not affected but the stability of the tripeptide in the presence of an oxidizing agent was diminished.     

Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g_av. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu²⁺, Zn²⁺ and Hg²⁺ inhibited, whereas Mg²⁺, Mn²⁺ and Fe²⁺ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Separation of β-N-acetylglucosaminidase isoenzymes from liver and plasma of six mammalian species by DEAE-cellulose chromatography

• 1.1. The β-N-acetylglucosaminidase (EC 3.2.1.30) activities of human, pig, calf, lamb, rat and rabbit liver and plasma have been investigated.
• 2.2. All preparations had maximum activity between pH 4.0 and 4.5 and K_m values with the substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside ranged from 0.54 to 2.54 mM.
• 3.3. The isoenzyme profiles of liver and plasma β-N-acetylglucosaminidase activity were compared using DEAE-cellulose chromatography. In all species the major anionic component of liver (β-N-acetylglucosaminidase A) was eluted at a higher salt concentration than the most anionic plasma isoenzyme.
• 4.4. The plasma β-N-acetylglucosaminidase A isoenzyme of all species contained sialic acid residues whereas only the rabbit, pig and calf liver isoenzymes were sialylated.

     

Effect of pH on the inhibition of the nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from baker''s yeast by anions

1. The sensitivity of the NAD(+)-specific isocitrate dehydrogenase from baker's yeast towards inhibition by anions decreases with decrease in pH. The patterns of the pH-dependence of the enzymic activity can be explained by this effect. 2. In the presence of a high isocitrate concentration, citrate, unlike AMP, has no antagonizing effect on the inhibition of the enzyme by anions. In the presence of AMP, citrate inhibits the enzyme at high isocitrate concentration and activates at low isocitrate concentration. 3. The effects on the enzymic activity of the previous incubation of the enzyme were studied in relation to the substrate concentration, the chloride concentration and the presence of citrate and AMP.     

Purification and some properties of cytosol 5′-nucleotidase from rat liver

A 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was highly purified from rat liver. The preparation appeared homogeneous on the criteria of disc-gel electrophoresis.A pH optimum at about 6.5 was observed for all substrates tested. The activity of this enzyme was absolutely dependent on the presence of various bivalent metal salts. The highest V value was attained with MgCl₂ and the concentration at half-enzyme saturation was lowest with MnCl₂. The enzyme had markedly higher affinities for IMP, dIMP, GMP and dGMP than the other 5′-mononucleotides, although V values for all the substrates tested were in the same order of magnitude.The activity of this enzyme was stimulated by various alkali metal salts, some carboxylic acids and adenine nucleotides. When AMP was used as substrate, the substrate-velocity plot was sigmoidal and NaCl, Tris-maleate and ATP stimulated the enzyme by decreasing the sigmoidicity of the plot. When IMP was used as substrate, the substrate-velocity plot was hyperbolic and these three activators stimulated the enzyme by increasing the V and decreasing the K_m value.Some of these results provided consistent evidence for the identity of this enzyme and the cytosol 5′-nucleotidase, the presence of which had been reported in crude preparations from rat liver.     

Solubilization of 17β-hydroxysteroid dehydrogenase from rat liver microsomes

The conditions for the solubilization of 17β-hydroxysteroid dehydrogenase from a rat liver microsomal preparation with the non-ionic detergent Triton X-100 were studied. The recoveries of 17β-hydroxysteroid dehydrogenase activity and of proteins in the solubilized form were determined as a function of detergent concentration, of pH and temperature, of incubation time and of saline concentration. The soluble fraction obtained under the optimal conditions contained 80% of the proteins and 75% of the enzymatic activity of initial microsomes. The presence of Triton X-100 in the solubilized proteins was not essential for enzyme activity.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Mold

A mold which produced α-galactosidase and little invertase was isolated and identified as Mortierella vinacea. α-Galactosidase formation of the mold was induced by galactose, melibiose, raffinose and lactose. Among these inducers lactose showed the most stimulative effect. α-Galactosidase was produced by either Koji method or submerged culture method, but in the latter most α-galactosidase was found in the mycelium fraction.

Hydrolysis of raffinose in beet molasses was studied with the α-galactosidase in the mycelium fraction and about 80% of raffinose was found to be hydrolyzed by the enzyme preparation.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates

The objective of this paper was the investigation of a suitable Sepabeads^? support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads^? EC-EA and Sepabeads^? EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads^? EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.     

Incorporation of cytosol δ-aminolevulinate synthase of rat liver into the mitochondria in vitro

When ¹²⁵I-labeled cytosol δ-aminolevulinate synthase was incubated in suspensions of rat liver mitochondria, the enzyme was incorporated into the mitochondira at the rate that was linear with time and with the [¹²⁵I]enzyme added. Subfractionation of the mitochondria using a digitonin technique revealed that the [¹²⁵I]enzyme was incorporated into the innermembrane-matrix fraction where endogeneous δ-aminolevulinate synthase is located.     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Studies on the heterogeneity of the 5′ ends of the protamine mRNAs from rainbow trout testis

The structures of the 5' termini of the protamine mRNAs (PmRNAs) have been investigated by inhibiting their translation in wheat-germ extracts in the presence of 7-methyl guanosine 5'-phosphate (m⁷GMP), an analogue of cap structure in mRNAs. Second, the cap structures on PmRNAs were examined by labelling the RNA at the 5' end with T₄ polynucleotide kinase and [-³²p]ATP before and after removal of these structures with tobacco acid pyrophosphatase and alkaline phosphatase. The results indicate that cap structures of the PmRNAs are heterogeneous. It appears that the mRNAs coding Ior protamine components C_I and C_III have at least a cap 1 structure while the mRNAs coding for C_II do not appear to be capped or methylated.     

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

A procedure is described for the preparation of extensively purified β-d-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the β-glucosidase in the high speed supernatant (100 000 × 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. β-Glucosidase activity co-chromatographs with β-d-galactosidase, β-d-fucosidase, α-l-arabinosidase and β-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 β-glucosidase, respectively. The specific activity of the apparently homogeneous β-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-β-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 (R_F, 0.67). β-Glucosidase activity is inhibited competitively by glucono-(1 → 5)-lactone (K_I, 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 β-d-glucose, it will not hydrolyze xylosyl-O-serine, β-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 β-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convulated tubule. The enzyme is also present in relatively large ammounts in the villus cells, but not crypt cells, of the intestine. the physiological subtrates and function of the enzyme are unknown.