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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.

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.

Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116,000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phosphate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.

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.

The effect of aflatoxin B1 on lipids of liver, kidney, adipose and plasma of rats was studied. A single dose administration (6 mg/kg body weight) increased liver and kidney weights and their total lipids within 24 h. Increase in liver lipids was confined mainly to phospholipid and cholesterol, whereas triglycerides showed a significant decrease. Adipose tissue triglycerides were, however, increased. Plasma showed decreases in triglycerides, free fatty acids and cholesterol. Incorporation studies with palmitate-1-14C revealed increased incorporation in adipose tissue lipids and decreased incorporation in liver and plasma lipids, thereby indicating an increased synthesis of lipids in adipose. Their mobilization to plasma was, however, inhibited, hence the low levels of triglyceride in liver. But the adrenals showed hypo-activity upon aflatoxin B1 administration.

Phospholipids of Mycobacterium phlei ATCC 354 and Mycobacterium smegmatis CDC 46 consist of cardiolipin, phosphatidyl ethanolamine, tri-acylated dimannophosphoinositide, tetra-acylated dimannophosphoinositide and tetra-acylated pentamannosphosphoinositide. A comparative study of lipid patterns of M. phlei ATCC 354 and of M. smegmatis CDC 46 in relation to age of culture revealed higher total lipid level and increased activity of malate-vitamin K reductase, a phospholipid requiring enzyme, during the early logarithmic growth phase of the former. No appreciable change occurred in the latter. The high total lipid content coincides with an increase in phospholipid, brought about apparently by the increase in malate-vitamin K reductase. Changes in cardiolipin and phosphatidyl ethanolamine appeared to be unique to M. phlei ATCC 354. However, in both bacterial species, a decrease in glyceride and a progressive increase in tuberculostearic acid with a concomitant decrease in oleic acid, occurred with ageing.

Radiorespirometric studies using glucose labelled at 1, 2, 3-4, and 6 positions and enzymatic studies were conducted to determine the primary pathways of glucose dissimilation in Mycobacterium tuberculosis H37Rv. The pattern of 14CO2 recovery was C3-4 greater than C1 greater than C6 = C2. The Embden-Meyerhof pathway was found to be the predominant pathway for glucose oxidation, operative to the extent of 94%. The pentose phosphate pathway accounted for the remaining 6%. Maximum incorporation of 14C into cellular components was from C2 and C6 labelled glucose.

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.

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.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest Km value. The enzyme showed an absolute requirement for divalent cations (either Mg2+ or Mn2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg2+-activated enzyme to varying degrees (Ni2+ greater than Zn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+). The differences in the kinetic responses of the enzyme to Mg2+ and Mn2+ are discussed.

A Mg+2-(Ca+2)-activated adenosine triphosphatase activity has been demonstrated in saprophytic, human pathogenic and non-pathogenic, and atypical, species of mycobacteria. (Na+ + K+)-activated adenosine triphosphatase was absent in the species investigated. The effect of age of culture on enzyme activity was revealed in an increase up to mid logarithmic phase of growth, and a decline thereafter. Differences in oxygen tensin during growth did not alter enzyme activity. Isonicotinic acid hydrazide and streptomycin inhibited the enzyme activity.

The effect of zinc and phytic acid on [1-14C]-acetate incorporation into aflatoxins by resting mycelium was studied. When different levels of ZnSO4 were used to study its effect on the incorporation of [1-14C]-acetate into aflatoxins, it was found that the specific radioactivity incorporation into aflatoxins was maximum at the level of 10 mM-ZnSO4. At this concentration the change in the specific radioactivities of aflatoxins B1 + B2 and aflatoxins G1 + G2 were +74.61% and +29.66%, respectively. On the other hand, phytic acid had an inhibitory effect on the incorporation of [1-14C]-acetate. These observations have been correlated in order to find out why soyabean is unable to produce aflatoxins by Aspergillus parasiticus.

Effect of excess feeding and depletion of vitamin A on the ability of adipose tissue to maintain plasma free fatty acid levels has been studied in rats. Both in hypervitaminosis A (fed 9 mg of retinol for 2 consecutive days) and in vitamin A deficiency (kept on a vitamin A-deficient diet for 6 weeks) the rats showed elevated levels of plasma free fatty acids. Hypervitaminosis A caused a decrease in the fatty acid release from adipose tissue on in vitro incubation, probably due to lowered levels of cyclic AMP. On the other hand, adipose tissue from vitamin A-deficient animals showed an increased lipolytic rate as compared to that of the controls. No change in the lipogenic ability was indicated in either of the conditions as indicated by the activities of enzymes involved in this process. We conclude that the fatty acid homeostasis can be greatly influenced by the vitamin A status of the animals.

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-14C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.

The effect of different combinations of aflatoxin B1, alpha-amanitin and actinomycin-D on the incorporation of orotic acid-6-14C into RNA was investigated using rat liver slices. The results support the view that the mode of action of aflatoxin B1 and alpha-amanitin are similar in some respects, and that aflatoxin B1 and actinomycin-D act according to different mechanisms. Evidence was also obtained about the formation of an active metabolite of aflatoxin B1 in this system.

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.

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.

Uptake of aflatoxin [14C]-B1 by the liver and kidney slices of different animal species clearly indicates that the transport of the toxin to different organs of the respective species is not the major cause for the reported variation in the species susceptibility to aflatoxin.

2-Mercaptoethanol inhibits growth of Aspergillus parasiticus NRRL 3240 and aflatoxin formation by the fungus. When added to the resuspended medium, 2-mercaptoethanol inhibited [1-14C]acetate incorporation into both aflatoxins and neutral lipids, thereby showing that it acts at an early stage of aflatoxin biosynthesis. The inhibition is probably due to its chelating action on zinc, which is essential for aflatoxin production. It is proposed that any chelating agent that selectively binds to zinc will inhibit aflatoxin formation.