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The phospholipid-associated protein (55-67 kDa) fraction of Mycobacterium tuberculosis H37Rv was purified as the DE-V protein fraction. This DE-V fraction was used for diagnosis of tuberculosis by enzyme-linked immunosorbent assay (ELISA), detecting IgG antibody in sera collected from different categories of tuberculosis patients, i.e. with acid fast bacilli (AFB) culture-positive pulmonary tuberculosis, with AFB culture-negative, but radiologically suspected, pulmonary tuberculosis, extrapulmonary tuberculosis, and control groups of patients suffering from diseases other than tuberculosis (asthma and/or rhinitis, lepromatous leprosy) as well as from healthy volunteers. Encouraging operational ELISA validity could be achieved with 93% sensitivity, 100% specificity, 97% efficiency, 100% positive predictivity and 95% negative predictability even among the extrapulmonary and suspected pulmonary tuberculosis patients. The above assay was insensitive but with 100% specificity among control group of patients suffering from diseases other than tuberculosis. The DE-V protein fraction was associated with phosphatidyl inositol and phosphatidyl inositol mannosides. The dissociation of phospholipid-protein complex decreased ELISA specificity. ELISA reactivity of the DE-V fraction appeared to be thermostable; thus, it may have serodiagnostic utility in developing countries.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.

With a view to detect specific M. tuberculosis infection, mycobacterial proteins were purified initially by ammonium sulphate precipitation followed by ion-exchange chromatography. Out of the three fractions, namely P1, P2 and P3 precipitated with increasing concentrations (0-25%, 26-65% and 66-100% respectively) of ammonium sulphate, P2 fraction was found to be more immunoreactive. P2 fraction proteins were further fractionated into five fractions by salt gradient using DEAE-cellulose DE-52 ion-exchange chromatography. Immunoreactivity against tuberculous patients sera of all the fractions was assessed using ELISA test. The last fraction (DE-V) eluted with high salt concentration was found to have a more specific immunoreactive set of proteins within the range of 55 kD to 67 kD molecular weight. Multiple non-specific proteins were distributed in all the other fractions. ELISA test using P2 fraction proteins against tuberculous patients sera showed significantly higher (p less than 0.01) titre even in the absence of any other bacteriological evidence. DE-V fraction of P2 proteins was found to have a significantly high specificity for detecting M. tuberculosis infection in clinically confirmed and suspected tuberculous patients indicating its application in the diagnosis of the disease.

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.

The breakdown and renewal of phospholipids and glycerides from spheroplasts of Mycobacterium smegmatis ATCC 14468 were studied by means of pulse labelling techniques using 1-14C-palmitate. Most of the radioactivity incorporated into total lipids was recovered from phospholipids. Among the phospholipids studied, cardiolipin had the highest radioactivity and specific activity, suggesting its higher pool size as compared to other fractions. The appreciable loss of radioactivity for cardiolipin in pulse chase experiments within growing cells suggests its metabolically active and dynamic nature. Glycerides exhibited no appreciable turnover rates.

Adenosine triphosphate (ATP) was found to inhibit the incorporation of labelled acetate into aflatoxins while adenosine diphosphate (ADP) and adenosine monophosphate (AMP) stimulated the process. Exogenous cyclic AMP and its derivative, N6-O2' -dibutyryl cyclic AMP produced efficient (1-14C)-acetate incorporation into aflatoxins at all the concentrations tried (0.1, 0.5 and 1 mM). The stimulation of incorporation of labelled acetate into aflatoxins was significant at 0.1 and 1 mM concentration of the cyclic nucleotide but was found to be maximum at 0.5 mM concentration.

Young, weanling rats fed wheat as a sole source of protein have been shown to develop periportal liver lipid infiltration. An attempt has been made to study the mechanism by which these lipid changes are produced. Weanling, male albino rats were fed three diets containing 10% protein based on wheat, wheat fortified with 0.2% lysine or casein, as control. Plasma and liver lipids, incorporation of [1-(14)C]acetate and [U-14C]glucose into hepatic lipids and triglycerides were studied after 6 weeks of feeding. Results of radioactive incorporation studies indicated that hepatic lipogenesis was depressed in rats fed wheat diets. However, adrenal cortical function as well as increased mobilization of lipids from adipose tissue was observed in these rats. Impaired secretion of lipids from liver to plasma is another factor which may be responsible for hepatic lipid accumulation in rats fed the lysine-deficient diet. Rats fed the lysine fortified wheat diet represented a more normal metabolic state though some features of protein deficiency were evident in this group too.

Evidence for the synthesis of insoluble peptidoglycan by spheroplasts of Mycobacterium smegmatis ATCC-14468 was obtained by inhibition of synthesis by vancomycin and cycloserine and no inhibition of synthesis by tetracycline. Radioactivity in the peptidoglycan fraction decreased when treated with lysozyme. The ability of spheroplasts to synthesize macromolecular peptidoglycan was half that of whole cells. A relationship was obtained between the amount of intact cell wall present and peptidoglycan synthesized: as the amount of intact cell wall decreased, the ability to synthesize macromolecular peptidoglycan also decreased.

Lungs of rats intoxicated by paraquat either due to parenteral administration or incubation of tissue slices with toxin appear to be responsive to the action of polyamines. Putrescine spermidine and spermine were able to significantly counter paraquat-induced augmentation of lipid peroxidation and superoxide dismutase (SOD) activity in the lungs of rats. Non-physiological polyamines like cadaverine and agmatine were also found to diminish peroxidation of lipids as well as SOD activity stimulated by paraquat. The protective action of polyamines could be due to membrane stabilization and prevention from oxidant damage.

The role of zinc on the primary metabolism of Aspergillus parasiticus in relation to aflatoxin biosynthesis was studied. Zinc deficiency impaired growth and metabolism of nucleic acid and protein while stimulating that of lipid. The activities of enzymes of glycolytic cycle were affected indicating that these are zinc dependent enzymes. Impaired glycolytic cycle tends to reduce the level of primary metabolites like pyruvate, citrate and oxaloacetate which trigger aflatoxin formation. During stationary phase these precursors accumulate as these are not used up for the synthesis of secondary metabolites (aflatoxins) due to zinc deficiency. ATP and energy-charge (E.C.) which are important for various physiological processes like growth are reduced in zinc deficiency. Also zinc deficiency results in low levels of AMP which favour synthesis of lipid. Inorganic phosphate accumulated due to zinc deficiency and this may be unfavourable for aflatoxin synthesis.

Irradiation with ultraviolet light (360 nm) of cell-free extracts, electron-transport particles, and soluble components from Mycobacterium phlei resulted in the loss of malate oxidation by the flavine adenine dinucleotide pathway both in cell-free extracts and reconstituted systems. Addition of vitamin K1 restored the loss to the extent of 14% and 11% in cell-free extracts and reconstituted systems respectively. Electron-transport particles from M. phlei upon reduction with malate exhibited electron-paramagnetic resonance signals at g = 2.002 and 1.94, characteristic of napthosemiquinone and nonheme iron protein, respectively. Upon irradiating the particles with ultraviolet light (360 nm) these signals were not observed. Particulate flavine-adenine-dinucleotide-dependent malate dehydrogenase (EC 1.1.1.37) of M. phlei assayed by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide and phenazine methosulfate - 2,6-dichlorophenolindophenol systems, which trap electrons at cytochrome c and at the flavine level respectively, was inhibited by o-phenanthroline. These observations suggest that nonheme iron protein is sensitive to ultraviolet light (360 nm) and participates before or in combination with flavine in the malate (flavine adenine dinucleotide) pathway of M. phlei.

Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.

Exposure of male albino rats in the weight range of 70-200 g to 25,000 ft of simulated altitude for 6 h at 32 degrees C caused an increase in plasma free fatty acids with decreased oxidation of palmitic acid-1-14C in lung slices of hypoxic rats. The in vivo esterification of palmitic acid-1-14C to form truglycerides, phosphatidylcholine and phosphatidylethanolamine was also low indicating decreased utilization of fatty acids by hypoxic lungs. De novo lipogenesis was observed to decreased to decrease because of decreased fatty acid biosynthesis as judged from acetate---14C incorporation. The specific activities of triglycerides, phosphatidylcholine and free cholesterol were also decreased. The decreased incorporation into phosphatidylcholine affected the incorporation into phosphatidylcholine of alveolar surfactant (2,000 g sediment fraction). The results are discussed in view of possible decreased energy status of hypoxic lungs.

The effect of a single oral dose of dieldrin (30 mg/kg body weight) on lipid metabolism in rats was studied. Liver lipids content increased and this increase was mainly in the triglyceride fraction. The incorporation of acetate-14C into fatty acids was decreased indicating an inhibition of lipogenesis. Fatty acid oxidation was increased. Palmitate-14C incorporation into the triglyceride fraction was enhanced pointing to an overall increased utilization of fatty acids.

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.

The role of blastospores in the protection of Aspergillus parasiticus from high levels of aflatoxins was studied. The strain protects itself from aflatoxicity by forming thick-walled blastospores. The formation of blastospores was not observed under conditions of reduced aflatoxin formation, e.g., under zinc and asparagine deficiencies. The germination of blastospores coincided with an increase in the specific activity of glutamate dehydrogenase (NADP) and a simultaneous decrease in the specific aflatoxin production.

Pyrimidine biosynthesis and its regulation in the presence of different carbon and nitrogen sources in the growth medium of Mycobacterium smegmatis were studied. M. smegmatis TMC 1546 cells grown in shake culture were found to have marginally higher pyrimidine enzyme biosynthesis activities than cells grown in static culture. The activity was highest at the mid-log phase of growth during both surface and shake cultures, suggesting that the cells were metabolically most active at this stage of growth. Replacement of glycerol by glucose and fructose slightly increased the activities of carbamyl phosphate synthetase and aspartate transcarbamylase. The enzyme activities involved in pyrimidine biosynthesis decreased when citrate was replaced by succinate, fumarate, pyruvate or acetate in the growth medium. The activities of the enzymes in pyrimidine biosynthesis were found to decrease when asparagine as a nitrogen source in the medium was replaced by glutamate, glutamine or ammonium chloride. The presence of ornithine in place of asparagine in the growth medium increased these enzyme activities, while the presence of arginine instead of asparagine in the growth medium decreased these enzyme activities, though the differences in activity were small. The activity of aspartate transcarbamylase in vitro was inhibited by arginine. In the case of cells grown in the presence of ornithine, the activity of aspartate transcarbamylase was induced by ornithine, but inhibited by arginine.

Glucose metabolism in Mycobacterium smegmatis was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Glucose is oxidized in this organism mainly through the Embden-Meyerhof-Parnas pathway, irrespective of the carbon source used for growth. The pentose phosphate pathway plays only a minor role and its extent depends on the carbon source used for growth. Enzymes of glycolytic and oxidative pathways were detected in cells grown on glucose, glycerol, or pyruvate but enzymes of the Entner-Duodroff pathway could be detected only in glucose-grown cells. Labeled acetate is utilized by cells cultured on glucose, glycerol, and pyruvate. In all cases more of C1 of acetate was converted to CO2 while incorporation into cellular constituents was maximum from C2 of acetate.

Ca2+-sensitivity of sheep lung cyclic-3',5'-nucleotide phosphodiesterase is provided by endogenous tightly bound calmodulin. The calcium sensitivity of a highly purified enzyme was desensitized by increasing the assay temperature. It could also be desensitized to Ca2+-activation by thiols such as dithiothreitol. The thiol-induced desensitization could be partially reversed by dialysis and almost completely reversed by dilution. The results presented in this paper indicate that thiols are possibly involved in the interaction of calmodulin with cyclic-3',5'-nucleotide phosphodiesterase. This is the first report on temperature and thiol-induced desensitization of Ca2+-sensitivity of a cyclic-3',5'-nucleotide phosphodiesterase.

The presence of adenosine-3',5'-monophosphate (cAMP), adenylate cyclase and the effect of glucose on cAMP levels in Mycobacterium spp have been reported earlier. To understand the role(s) of cAMP in these organisms, the induction of various enzyme systems was studied. Beta-galactosidase and L-tryptophanase were present at low levels of activity and could not be induced. Glycerokinase was inducible but the induction was not affected by glucose. The fructose uptake system was inducible, and the induction was lowered in the presence of glucose, but cAMP could not reverse the inhibition. cAMP binding protein was not detectable under a variety of conditions. On the basis of the lack of active cAMP binding protein, a model has been proposed to explain the apparent lack of expression of cAMP function in Mycobacterium smegmatis CDC 46.

Several derivatives of coumarin inhibited mitochondrial respiration and ATPase activity. The extent of inhibition depended on the concentration of the coumarins as well as on the substituents of the coumarin ring. Some of the coumarins stimulated ATPase activity, but all of them inhibited uncoupler-stimulated ATPase activity. Coumarins with free or substituted phenolic groups were found to exert profound effects on respiration and ATPase activity.

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.

Cyclic guanosine 3'5'-monophosphate was isolated and purified from Mycobacterium smegmatis TMC 1515 using ion exchange chromatography. It was characterized by thin layer chromatography and radioimmunoassay. Guanylate cyclase and cGMP phosphodiesterase were detected in the cytosolic and particulate (37,000 X g pellet) fractions respectively. On the basis of our observations, cGMP appears to play a dual role (i) at the time of induction of cell proliferation and (ii) protects the bacteria against unfavourable surroundings during stationary phase of the growth. This is the first report demonstrating presence of cGMP, guanylate cyclase and cGMP phosphodiesterase in mycobacteria.

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) has been purified from Mycobacterium smegmatis CDC 46 using (NH4)2SO4 precipitation, negative adsorption on DEAE-cellulose, 2',5'-ADP-Sepharose affinity chromatography and Sephadex G-200. The enzyme was purified 1041.6-fold and the preparation was found to be homogeneous on column chromatography, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. Alanine and threonine were identified as the N- and C-terminal amino acids of glutamate dehydrogenase from M. smegmastis. The enzyme kinetics and regulation of glutamate dehydrogenase activity by different nutritional factors has been studied. Initial velocity plots showed that the reaction mechanism of glutamate dehydrogenase from M. smegmatis followed an ordered sequential ter-bi mechanism.

The effect of a single interaperitoneal injection (6 mg/kg body weight) of aflatoxin B1 in propylene glycol on pyridine nucleotides and NDP linked dehydrogenases was studied 24 h after administration of the toxin. The liver showed a decrease in total proteins and pyridine nucleotides though levels of NADP and NADPH remained unchanged. Levels of NAD and NADH were decreased. The activities of hepatic of hwpRIX of hepatic malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) were not altered though ICDH showed an increase when expressed on protein basis. However, there was a significance decrease in the activity of combined HMP dehydrogenases. Adipose tissue showed increased activities of the HMP dehydrogenasess.