Purification and some properties of apurinic/apyrimidinic dna endonucleases in rat liver

• 1.1. Three kinds of apurinic/apyrimidinic (AP) DNA endonucleases, APcI, APcII, APcIII were purified from rat liver chromatin.
• 2.2. Molecular weights of APcI, APcII and APcIII were 30,000, 42,000 and 13,000 Da, which have isoelectric points of 7.2, 6.3 and 6.2, respectively.
• 3.3. Mg²⁺ was essential for the activities of these 3 enzymes, and sulfhydryl compounds (βercaptoethanol) had a stimulatory effect on the enzyme activities while N-ethylmaleimide and HgCl₂ inhibited the enzyme activity.
• 4.4. K_m values of APcI, APcII and APcIII for AP site of DNA were 0.53, 0.27 and 0.36 μM, respectively, and AMP was the most potent inhibitor to these three enzymes among nucleotides tested.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.

     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

• 1.1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme.
• 2.2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities.
• 3.3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long.
• 4.4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature.
• 5.5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K.
• 6.6. The enzyme is subject to substrate inhibition and the K_m value is 159 × 10⁻³mM DNA-P.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

• 1.1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity.
• 2.2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form).
• 3.3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions.
• 4.4. According to V_max/K_m values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate.
• 5.5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.