Meir Lev

The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Partial purification and properties

Abstract The first enzyme of the sphingolipid pathway has been purified >100 times. The enzyme has an apparent M r of 66,500. The partially purified enzyme was used to determine K m for serine (1.13 m m ); the apparent K m for palmitoyl CoA was anomalous. The pH optimum of the synthetase was found between pH 7.0 and 7.6. The effectiveness of various acyl CoAs was related to chain length showing a symmetrical distribution around C 16 (palmitoyl). Decanoyl CoA was the shortest compound showing activity. The unsaturated C 18 , (oleoyl) CoA, showed significantly reduced activity as compared with stearoyl CoA. A number of compounds which inhibited synthetase activity were examined; cysteine competitively inhibited the response of the enzyme to serine ( K i = 0.49 mM) and a number of halogens were inhibitory in the order I = Br > Cl > F. Thiocyanate was a potent inhibitor of 3KDS synthetase (70% inhibition at 1 m m ). The response of the apoenzyme to its cofactor pyridoxal phosphate was linear from 0 → 10 μ m and reached a maximum between 10 and 20 μ m . In extracts from mid-log-phase cells, approximately 50% of the synthetase exists in the apoenzyme form.

The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Induction by vitamin K☆

Abstract An enzyme present in cell-free extracts of B. melaninogenicus grown with vitamin K is described which catalyzes the synthesis of 3-ketodihydrosphingosine from palmitoyl CoA and l -serine. Activity of the 3-ketodihydrosphingosine synthetase was measured as a function of time, palmitoyl CoA concentration, and serine concentration. The Bacteroides synthetase differs from corresponding enzymes from brain microsomes and from yeast in that it is present in the 100,000g supernatant of sonicated cells and is not associated with any particulate fraction. Extracts prepared from cells depleted of vitamin K showed only slight 3-ketodihydrosphingosine synthetase activity. Neither vitamin K1, menadione, nor pyridoxal phosphate were effective in enhancing the activity in cell-free extracts of vitamin K-depleted B. melaninogenicus. However, induction of the enzyme activity in intact cells was demonstrated by the addition of vitamin K to a vitamin K-depleted culture. Synthetase activity was found to be increased 15 min following the addition of the vitamin, reached a maximum at 75 min, and thereafter remained constant. Both puromycin and rifampcin inhibit induction of the enzyme by vitamin K1 suggesting that vitamin K induces de novo synthesis of the synthetase.

AN ANTIBODY AGAINST REVERTANT FORMS OF CELL-WALL-DEFICIENT BACTERIAL VARIANT IN SERA FROM PATIENTS WITH CROHN'S DISEASE

Sera from patients with Crohns disease or ulcerative colitis, and from controls were examined by indirect immunofluorescence for antibody against two strains of pseudomonas-like cell-wall-defective bacterial variants. Serum samples from 22 of 25 patients with Crohns disease produced fluorescence of both revertant cell-wall-defective bacterial strains. Intensity of fluorescence correlated positively with the degree of disease activity. Sera from 23 patients with ulcerative colitis and from 15 control subjects did not produce any significant staining of either of the two revertant cell-wall-defective bacterial strains. Absorption of sera with Escherichia coli, Bacteroides thetaiotaomicron, and Pseudomonas aeruginosa did not alter the intensity of fluorescence in patients with Crohns disease, whereas similar absorption of sera from patients with ulcerative colitis and controls abolished the slight staining of cell-wall-defective strains produced by 29% of unabsorbed serum samples.

INFLUENCE OF MICROORGANISMS ON MAMMALIAN METABOLISM AND NUTRITION, WITH SPECIFIC REFERENCE TO OXYGEN CONSUMPTION, CARBON DIOXIDE PRODUCTION, AND COLONIC TEMPERATURE*

For a number of years, we have been attempting to delineate the specific nature and significance of the complex influences of microorganisms on mammalian metabolic and nutritional processes. An idea of the variability of these influences is evident when one considers the effects of the ordinary microflora present in healthy laboratory animals on their responses to various nutritional deficiencies. The reported effects range from accentuation of the deficiency (e.g. vitamin C ) l to amelioration of the deficiency, (e.g. vitamin K ) 2 and, in some cases, there are no major effects (e.g. vitamin A ) .3 We began investigating the effects of intestinal bacteria on the oxygen consumption, carbon dioxide production, and body temperature of the rat because of the striking differences in body composition, fecal nitrogen excretion, and response to food deprivation we had noted in germfrees. conventional, and conventionalized rat^.^,^ Conventionalized rats, according to our terminology, are animals which are germfree until weaning, then purposefully contaminated with cecal contents from open-animal room rats thereby providing a mixed microflora. These multicontaminated rats are then maintained in the identical type of isolators used for germfree rats. In our usual experiment, germfree litters are divided at weaning in two groups, one of which is kept germfree while the other is conventionalized. By this type of control, the two groups of animals differ only in the presence or absence of microorganisms diet, housing, handling, air flow, air temperature and humidity, stimulation, etc., are the same. Rats purposefully contaminated with one or more specific bacterial species are handled in a similar fashion.

Dihydrosphingosine growth inhibition and repression of 3-ketodihydrosphingosine synthetase activity in Bacteroidesmelaninogenicus

Abstract Addition of dihydrosphingosine or 3-ketodihydrosphingosine to growing cultures of Bacteroides melaninogenicus markedly reduced the activity of 3-ketodihydrosphingosine synthetase in extracts. Neither compound reduced the activity of a previously solubilized preparation of the enzyme. Dihydrosphingosine and synthetic acetyl ceramides inhibited the growth of the microorganism; 3-ketodihydrosphingosine was not inhibitory and reversed the growth inhibition caused by dihydrosphingosine. This reversal may indicate a preferential utilization of 3-ketodihydrosphingosine over dihydrosphingosine by the cells for the biosynthesis of complex sphingolipids.

Role of nucleosides, 5-phosphoribosyl-1-pyrophosphate, and ribose 1-phosphate in the biosynthesis of phosphosphingolipids in Bacteroides melaninogenicus

Abstract Using a deenergized spheroplast system from Bacterioides melaninogenicus , the sphingolipid precursor 3-ketodihydrosphingosine is not incorporated into the complete sphingolipids, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol unless supplied with glutamine, ATP, ADP, or AMP. Adenosine, inosine, and certain other nucleosides were as effective as ATP. Purine bases and ribose, however, were inactive in this system. 5-Phosphoribosyl-1-pyrophosphate and ribose 1-phosphate also stimulated conversion of 3-ketodihydrosphingosine into ceramide phosphorylethanolamine and ceramide phosphorylglycerol, whereas ribose 5-phosphate showed only slight activity. Hypoxanthine was the main product formed from inosine and adenosine but there was no evidence for nucleotide formation. Adenosine stimulated 32 P i incorporation into cell phospholipids indicating that ribose 1-phosphate, formed via purine nucleoside phosphorylase, could be the compound stimulating sphingolipid synthesis in this system.

Impaired water metabolism in germfree rats.

Summary Diuresis following gastric gavage of water was examined in germfree and conventional rats. Germfree rats began to excrete urine 1 hr later than the conventional group and the volume of urine excreted by the germfree animals was less than excreted by the conventional group. Saline excretion patterns were the same for the two groups of animals. When 4% glucose was administered subcutaneously, excretion patterns were similar to that obtained following gastric gavage of water except that the onset of excretion was delayed 2 hr in the germfree as compared with the conventional group.