Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

Steroidogenesis in the desensitized rat corpora-lutea

Earlier workers have observed that in the leydig cell desensitization brings results in addition to down regulation of receptors, in leisons in the steroidogenlc pathway. In the present study immature rats having heavily leutinized ovaries were given 50 iu hCG and the desenasitized CL removed 48h later were used. At that time no change in the 5 3MSD activity and CAMP binding activity(a measure of CAMP dependent protein kinase) was observed.Followlng desensitization however,l)a significent increase in phosphodiestrase activity,ii)a 50% reduction in total mitochondrial cholesterol level, iii)a significant reduction in its ability to utilize cholesterol or hydrolyse its ester and iv)a significant lowering(by 66%)in cholesterol side chain clean age activity(by measuring pregnanalone formed) was observed. Pregnanalone production was restored to normalcy if exogenous cholesterol was added to the mitohondrial preparation. The results suggest that luteal desensitization is due in addition to down regulation of LH receptors, to a marked reduction in available cholesterol pool in the mitochondrial compartment. The increase in phosphodiestrase activity, though probably a secondary effect,might effectively contribute to the overall reduction in the steroid out-put by increasing the catabolism of CAMP.(Aided by grants from ICMR,New Delhi and WHO, Geneva).

Homoagmatine from Lathyrus sativus seedlings

A new guanidino amine has been isolated from Lathyrus sativus seedlings and chararterized as homoagmatine on the basis of various physico-chemical criteria including IR spectrum and comparison with that chemically synthesized. Homoagmatine is accumulated in the embryos axis while its precursor, homoarginine, is lost from the cotyledons. However, there was a progressive increase in homoarginine content of the embryo axis during development. Since the amine content of the whole seedlings corresponded to nearly 20–25 % of net decrease in homoarginine levels, it is concluded that the catabolism of homoarginine through homoagmatine represents a major pathway of metabolism of the arnino acid.

Metabolic enzymes as potential drug targets in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be-identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum

Arginase from Lathyrus sativus

The seeds of Lathyrus sativus contain the unusual amino acid homoarginine. The possible breakdown of homoarginine to lysine and urea has been investigated with enzyme extracts prepared from the seedlings of L. sativus. The results indicate that there is no separate homoarginase enzyme but that the arginase present has about 5 per cent activity towards Image -homoarginine as compared to that obtained with Image -arginine. The enzyme does not show an absolute dependence on Mn2+ for activity and maximal activation of the enzyme has been realized with Fe3+. It is concluded that the breakdown of homoarginine through the urea cycle may only represent a minor pathway for the catabolism of this compound in this plant.

Metabolism of ubiquinone in relation to vitamin a status in the rat

1. 1. Biosynthetic experiments in vitro with slices of livers from normal and vitamin A-deficient rats confirmed that synthesis of ubiquinone did not increase in vitamin A deficiency. 2. 2. During development of deficiency of vitamin A in the rat, there was a definite increase in the synthesis of ubiquinone at the 10-days stage but this reverted to low, initial level by 20 days and after. 3. 3. Vitamin A analogues, 3-dehydroretinal, 5,6-monoepoxyretinal and retinoic acid, which supported growth have restored ubiquinone concentration to the normal levels and relieved the lowering in its catabolism. The biologically inert 5,8-monoepoxyretinal was the least active of the analogues tested. 4. 4. The concentration and synthesis of ubiquinone in the liver decreased under conditions of hypervitaminosis A. 5. 5. The experimental evidence does not support the hypothesis of inverse relationship between vitamin A and ubiquinone synthesis.

Effect of cold exposure on the metabolism of ubiquinone in the rat

1. Accumulation of ubiquinone in the livers of rats exposed to a cold environment was shown to be due to both decreased catabolism during the entire experimental period and increased synthesis during an intermediate stage (10–20 days). 2. The increased endogenous synthesis in the cold-exposed rats was eliminated when ubiquinone accumulated in the liver after exposure for 40 days (coinciding with cclimatization), or by absorption of the exogenous dietary supply, possibly by the mechanism of end-product regulation.

Biosynthesis of ubiquinone and ubichromenol in vitamin A-deficient rats

1. The concentrations of ubiquinone and ubichromenol increased in the livers, but not in the intestines and kidneys, of rats maintained on a diet deficient in vitamin 2. After short time intervals (e.g. 2 h) following administration of the tracer, incorporation of [2-14C]mevalonate into ubiquinone and ubichromenol in livers of vitamin A-deficient rats was lower than for normal animals; this was in contrast to later times (e.g. 72 h) when it was higher. 3. The “newly synthesized” ubiquinone in livers of vitamin A-deficient rats was distributed in all the cell fractions without specific localization. 4. Absorbed exogenous [14C]ubiquinone and [14C]ubichromenol were retained in the livers of vitamin A-deficient rats to a larger extent and for a longer time than in the normal animals. 5. The results suggest that the accumulation of ubiquinone and ubichromenol in the livers of vitamin A-deficient rats is due to lowered catabolism and not to increased rate of synthesis.

Analysis of the hierarchical structure of the B. subtilis transcriptional regulatory network

The transcriptional regulation of gene expression is orchestrated by complex networks of interacting genes. Increasing evidence indicates that these `transcriptional regulatory networks' (TRNs) in bacteria have an inherently hierarchical architecture, although the design principles and the specific advantages offered by this type of organization have not yet been fully elucidated. In this study, we focussed on the hierarchical structure of the TRN of the gram-positive bacterium Bacillus subtilis and performed a comparative analysis with the TRN of the gram-negative bacterium Escherichia coli. Using a graph-theoretic approach, we organized the transcription factors (TFs) and sigma-factors in the TRNs of B. subtilis and E. coli into three hierarchical levels (Top, Middle and Bottom) and studied several structural and functional properties across them. In addition to many similarities, we found also specific differences, explaining the majority of them with variations in the distribution of s-factors across the hierarchical levels in the two organisms. We then investigated the control of target metabolic genes by transcriptional regulators to characterize the differential regulation of three distinct metabolic subsystems (catabolism, anabolism and central energy metabolism). These results suggest that the hierarchical architecture that we observed in B. subtilis represents an effective organization of its TRN to achieve flexibility in response to a wide range of diverse stimuli.

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 angstrom resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of approximate to 5 angstrom from the -phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative S(N)2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the - and the -phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the -phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.

Unconjugated Bilirubin exerts Pro-Apoptotic Effect on Platelets via p38-MAPK activation

Thrombocytopenia is one of the most frequently observed secondary complications in many pathological conditions including liver diseases, where hyperbilirubinemia is very common. The present study sought to find the cause of thrombocytopenia in unconjugated hyperbilirubinemic conditions. Unconjugated bilirubin (UCB), an end-product of heme catabolism, is known to have pro-oxidative and cytotoxic effects at high serum concentration. We investigated the molecular mechanism underlying the pro-apoptotic effect of UCB on human platelets in vitro, and followed it up with studies in phenylhydrazine-induced hyperbilirubinemic rat model and hyperbilirubinemic human subjects. UCB is indeed found to significantly induce platelet apoptotic events including elevated endogenous reactive oxygen species generation, mitochondrial membrane depolarization, increased intracellular calcium levels, cardiolipin peroxidation and phosphatidylserine externalization (p < 0.001) as evident by FACS analysis. The immunoblots show the elevated levels of cytosolic cytochrome c and caspase activation in UCB-treated platelets. Further, UCB is found to induce mitochondrial ROS generation leading to p38 activation, followed by downstream activation of p53, ultimately resulting in altered expression of Bcl-2 and Bax proteins as evident from immunoblotting. All these parameters conclude that elevated unconjugated bilirubin causes thrombocytopenia by stimulating platelet apoptosis via mitochondrial ROS-induced p38 and p53 activation.