Effect of vitamins A and K on colon lysosomes

1. 1. Colon lysosome were separated by differential centrifugation and lysosomes with three different densities, probably arising from the three layers of colon, were found. 2. 2. Hypervitaminosis A resulted in a significant increase in prothrombin time which was restored to normal on vitamin K1 (20) supplementation. 3. 3. There was no appreciable change in the liver storage of vitamin A between hypervitaminotic rats receiving vitamin A and those rats receiving vitamin K1 (20) in addition to excess vitamin A. 4. 4. The colon lysosomes were unstable in hypervitaminosis A, showing an increased free activity of lysosomal enzymes like β-glucuronidase, acid phosphatase and arylsulphatase. This increase of free activity of lysoso3al enzymes in hypervitaminosis A could be prevented by oral supplementation of vitamin K1 (20). 5. 5. In "mild" vitamin A deficiency the release of arylsulphatase was significantly retarded, whereas the decreased free acid phosphatase activity was not significant. 6. 6. "Severe" vitamin A deficiency resulted in a significantly increased free activity of arylsulphatase and acid phosphatase, thus showing the instability of the lysosomal particles in this condition. 7. 7. Addition of vitamin K1 (20) to the incubation medium in vitro could prevent the vitamin A-induced release of arylsulphatase from liver lysosomes, whereas α-tocopherol was inactive. 8. 8. Retinol and retinoic acid were nearly twice as active as ethanol in the release of arylsulphatase from lysosomes in vitro, whereas 5,6-monoepoxyretinoic acid was inactive. 9. 9. The role of vitamins A and K on the lysosomal membrane structure is discussed.

Image -asparaginases from Mycobacterium tuberculosis strains H37Rv and H37Ra

M. tuberculosis H37Ra possesses two Image -asparaginases while the H37Rv strain possesses only a single enzyme. These enzymes have been purified and their properties studied. The two Image -asparaginases in H37Ra strain differ from each other in pH optima, heat inactivation, Michaelis constant and effects of inhibitors, while one of them resembles the single Image -asparaginase present in the H37Rv strain. Image -Cysteine inhibits both Image -asparaginases in an allosteric manner probably because it is one of the end-products in Image -asparagine metabolism. This is the first time that a qualitative difference has been reported in the enzyme pattern between the avirulent and virulent strains of M. tuberculosis.

Biosynthesis of isoleucine and valine in Mycobacterium tuberculosis H37 Rv

The enzymes involved in the biosynthesis of isoleucine and valine have been shown to be present in cell-free extracts of Mycobacterium tuberculosis H37Rv. In addition to the known enzymes of the pathway, cell-free extracts of this organism contain a new enzyme. When cell-free extracts were incubated with acetolactate and Image -ascorbic acid, without reduced nicotinamide adenine dinucleotide phosphate, the isomer of acetolactate, viz., α-keto-β-hydroxyisovalerate, was found to accumulate and was identified by different methods. The reaction is enzymic, and Image -ascorbic acid cannot be replaced by other reducing agents such as hydroquinone, 2,6-dichlorophenol indophenol, or glutathione; by derivatives of Image -ascorbic acid such as dehydroascorbic acid or dimethyl ascorbic acid; or by cobamide coenzyme. Since the extracts also isomerize α-acetohydroxybutyrate to α-keto-β-hydroxy-β-methylvalerate, the enzyme catalyzing the reaction has been termed “acetohydroxy acid isomerase.” This is the first time that the presence of acetohydroxy acid isomerase has been reported in any biological system and that a specific metabolic role has been assigned for Image -ascorbic acid. The extract also possesses reductase activity to convert α-keto-β-hydroxyisovalerate to α,β-dihydroxyisovalerate in the presence of reduced nicotinamide adenine dinucleotide phosphate.

Metabolism of Plasmodium berghei. I. Krebs cycle

The free parasites of Plasmodium berghei, obtained from infected cells of rats using an antiserum method, were investigated to study the operation of Krebs cycle. P. berghei was found to respire only with succinate; pyruvate, and other substrates of the Krebs cycle were not oxidized. The presence of a succinate dehydrogenase and a functioning cytochrome oxidase system was demonstrated. Cell-free extracts of free parasites showed the presence of enzymes for the utilization of C4 dicarboxylic acids; other enzymes of the Krebs cycle could not be detected. P. berghei differs from other species of Plasmodium in this respect.

Chemical modification of sheep plasma glycoprotein

Glycoprotein isolated from sheep plasma was chemically modified, and the effect of chemical modification on biological activities and immunological cross reactions has been studied. The removal of sialic acid resulted in a change in the “overall conformation” of the glycoprotein as evidenced by a decrease in viscosity of the glycoprotein solution and an increased susceptibility of the glycoprotein to proteolytic enzymes. Sialic acid-free glycoprotein no longer inhibited the tryptic activity or prolonged the clotting time of plasma. However, it could react with the antiserum to sheep plasma glycoprotein. The periodate oxidation of sheep plasma glycoprotein resulted in a complete loss of inhibition of trypsin activity, prolongation of plasma clotting time, and the ability to cross-react with the rabbit antiserum. The significance of periodate oxidation in relation to the possible sequence of sugars in the carbohydrate prosthetic group in the glycoprotein is discussed. Iodination and heating in buffers of acid and alkaline pH values of sheep plasma glycoprotein resulted in complete loss of trypsin activity and ability to prolong plasma clotting time. Iodination of the glycoprotein did not affect the immunological cross-reactivity.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid, the lathyrus sativus neurotoxin

The biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP) the Lathyrus sativus neurotoxin has been found to follow the scheme depicted below: {A figure is presented}. The first reaction is catalysed by oxalyl-CoA synthetase which has properties similar to that of the enzyme in peas. The second reaction is catalysed by another enzyme which is specific to L. sativus and is designated as oxalyl-CoA-α,β-diaminopropionic acid oxalyl transferase. The enzymes have been purified by about 60-fold and their properties studied. A partial resolution of the two enzyme activities has been achieved using CM-sephadex columns.

Intracellular distribution of coenzyme Q in rat liver

Coenzyme Q was found to be distributed in rat liver cell fractions. Mitochondria accounted for only 40–60% of the total. The presence of coenzyme Q in nuclei, isolated by several methods, could always be correlated with the presence of oxidative enzymes. It has been established that coenzyme Q is a constituent of microsomes. Administered coenzyme Q10-C14 was preferentially taken up by mitochondrial and microsomal fractions. Exogenous coenzyme Q appears to be rapidly metabolized.

Alkaline and acid phosphatases of the silkworm, Bombyx mori L.

The variations in the activities of the alkaline and acid phosphatases of the silkworm, Bombyx mori, were studied in all stages of the life cycle. From hatching until the spinning stage a steady increase was recorded in the activity of both the enzymes followed with a conspicuous decrease at each moult. During the pupal stage the alkaline phosphatase was almost absent, whereas the acid phosphatase maintained a high and constant value. Increase or decrease of the activity of the enzymes during larval development was reflected in a decrease or increase in the acid-soluble phosphorus content. Acid phosphatase activity slowly increased from laying of the eggs to hatching of the larvae with a concomitant decrease in the acid-soluble phosphorus. Tissue analysis showed a high concentration of the alkaline enzyme in the intestines, but the haemolymph was almost free of both enzymes. Feeding of inorganic phosphate increased the alkaline enzyme in the intestines, whereas glucose had no effect on either of the enzymes in the intestines.

High cholesterol diet and esterification of cholesterol by the intestinal mucosa of rats

Young male rats maintained on a diet containing 1% cholesterol were sacrificed at the end of 1st, 2nd, 3rd, 5th, and 7th week. Acetone powders prepared from their intestinal mucosa and pancreas were tested for the synthetic and hydrolytic activities for Vitamin A and cholesterol esters. The esterifying activity of the mucosal enzymes for both Vitamin A and cholesterol increased progressively up to the end of the 5th week; the increase in esterification of cholesterol was more marked with respect to saturated fatty acids, as compared to the unsaturated ones. The pancreatic enzymes remained unaffected. It is suggested that one of the reasons for the accumulation of cholesterol esters in animal tissues may be the increased esterification of the sterol in the mucosa induced by dietary cholesterol.

Interaction of heterocyclic thiols/thiones eliminated from cephalosporins with iodine and its biological implications

Hydrolysis of beta-lactam antibiotics by beta-lactamases (e. g., metallo-beta-lactamase, m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. It is shown in this paper that the thiol/thione moieties eliminated from certain cephalosporins by m beta l-mediated hydrolysis readily react with molecular iodine to produce ionic compounds having S-I bonds. While the reaction of MTT with iodine produced the corresponding disulfide, MDT and DMETT produced the charge-transfer complexes MDT-I-2 and DMETT-I-2, respectively. Addition of two equivalents of I-2 to MDT produced a novel cationic complex having an almost linear S-I+-S moiety and I-5(-) counter anion.However, this reaction appears to be highly solvent dependent. When the reaction of MDT with I2 was carried out in water, the reaction produced a monocation having I-5(-), indicating the reactivity of MDT toward I2 is very similar to that of the most commonly used antithyroid drug methimazole (MMI). In contrast to MMI, MDT and DMETT, the triazine-based compound MTDT acts as a weak donor toward iodine. (C)2010 Elsevier Ltd. All rights reserved.

Mechanism of hydroxylation of aromatic compounds: II. Evidence for the involvement of superoxide anions in enzymatic hydroxylations

The mechanism of hydroxylation reactions catalyzed by m-hydroxybenzoate-4-hydroxylase and anthranilate hydroxylase from Aspergillus niger was investigated using superoxide dismutase from ovine erythrocytes. Inclusion of superoxide dismutase in the assay mixtures of the two enzymes resulted in complete inhibition of the hydroxylation reaction, indicating the possible involvement of superoxide anions (O2−) in these reactions.

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.

Molecular Basis for the Role of Staphylococcus aureus Penicillin Binding Protein 4 in Antimicrobial Resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetyl-glucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. beta-Lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.