Regulation of sulfotransferase activity by vitamin K in mouse brain

We have shown previously that the administration of warfarin to 16-day-old mice results in a significant reduction in levels of sulfatides, and to a lesser degree a reduction of other sphingolipids in brain. Vitamin K stimulates biosynthesis of sulfatides in warfarin-treated mice. We now report that warfarin inhibits brain sulfotransferase activity. This inhibition is reversed by vitamin K. The treatment of normal mice with vitamin K stimulates the activities of sulfotransferase and arylsulfatase and the turnover rate of brain sulfatides. The ability of vitamin K to influence the activity of biosynthetic and catabolic enzymes and the turnover of sulfatides suggests a possible regulatory role for vitamin K in the maturing brain.     

Inhibition of cerebroside synthesis in the brains of mice treated with L-cycloserine

Subcutaneous injection of L-cycloserine resulted in a 28% reduction in cerebroside levels in mouse brain but had no effect on the levels of gangliosides. In contrast, intraperitoneal injection results in a reduction of ganglioside as well as cerebroside + sulfatide levels. The route of injection influenced the degree of 3-ketodihydrosphingosine synthase inhibition. Intraperitoneal injection caused a rapid decrease in synthase activity followed by recovery over 48 hr, whereas subcutaneous injection resulted in no inhibition over this time; only after daily injection for a week was synthase activity reduced 35%. One week following cessation of L-cycloserine administration, enzyme activity had recovered, whereas the cerebroside level continued to fall. All lipids and enzymes showed normal levels 3 weeks post-cycloserine administration. L-[3H]serine incorporation into glycolipids showed that cerebroside synthesis was most affected, whereas sulfatide synthesis was less affected. One week after cessation of cycloserine treatment, cerebroside synthesis was still severely inhibited, whereas sulfatide levels were near normal. Two weeks after cessation of L-cycloserine administration, synthesis of these glycolipids was similar to that of controls.     

Ineffectiveness of phylloquinone epoxide as an inhibitor of prothrombin synthesis in the rat

Phylloquinone epoxide (vitamin K₁-oxide), a metabolite of phylloquinone, does not inhibit prothrombin synthesis when administered in high doses to Sprague-Dawley and warfarin-resistant rats. Further, it does not accumulate to presumed inhibitory levels in the livers of rats given physiological doses of ³H-phylloquinone when they are anticoagulated with warfarin. These data do not support the Bell-Matschiner hypothesis that warfarin exerts its action by inhibiting the vitamin K oxide reductase which results in the accumulation of vitamin K oxide and the inhibition of vitamin K at its active site. Rather, our data support the view that vitamin K and warfarin combine at different sites with a single regulatory protein which serves as a conformational switch for prothrombin synthesis.     

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.     

Differential Effects of Dabigatran and Warfarin on Bone Volume and Structure in Rats with Normal Renal Function

Background

Warfarin, a widely used anticoagulant, is a vitamin K antagonist impairing the activity of vitamin K-dependent Bone Gla Protein (BGP or Osteocalcin) and Matrix Gla Protein (MGP). Because dabigatran, a new anticoagulant, has no effect on vitamin K metabolism, the aim of this study was to compare the impact of warfarin and dabigatran administration on bone structure and vascular calcification.

Methods

Rats with normal renal function received for 6 weeks warfarin, dabigatran or placebo. Bone was evaluated immuno-histochemically and hystomorphometrically after double labelling with declomycin and calcein. Aorta and iliac arteries were examined histologically.

Results

Histomorphometric analysis of femur and vertebrae showed significantly decreased bone volume and increased trabecular separation in rats treated with warfarin. Vertebra analysis showed that the trabecular number was higher in dabigatran treated rats. Osteoblast activity and resorption parameters were similar among groups, except for maximum erosion depth, which was higher in warfarin treated rats, suggesting a higher osteoclastic activity. Therefore, warfarin treatment was also associated with higher bone formation rate/bone surface and activation frequency. Warfarin treatment may cause an increased bone turnover characterized by increased remodelling cycles, with stronger osteoclast activity compared to the other groups. There were no differences among experimental groups in calcium deposition either in aortic or iliac arteries.

Conclusions

These findings suggest for the first time that dabigatran has a better bone safety profile than warfarin, as warfarin treatment affects bone by reducing trabecular size and structure, increasing turnover and reducing mineralization. These differences could potentially result in a lower incidence of fractures in dabigatran treated patients.     

Concurrent warfarin treatment further reduces bone mineral levels in 1,25-dihydroxyvitamin D3-treated rats

Weanling rats on a normal diet mobilized bone calcium in response to 11 daily injections of 125 ng of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)/100 g, body weight. This effect was most evident in the tibial midshaft, where calcium levels were reduced by 38% compared to untreated controls. Calcium levels were reduced by only 13% in the proximal tibial metaphysis, a region formed by longitudinal growth during the 11-day experiment. The concurrent daily administration of the vitamin K antagonist warfarin dramatically increased calcium mobilization from the tibial metaphysis of 1,25-(OH)2D3-treated rats. Compared to rats which received 1,25-(OH)2D3 alone, the calcium content of the tibial metaphysis in rats treated with 1,25-(OH)2D3 plus warfarin was reduced by 40.4% (p less than 0.001) and the total dry weight was reduced by 35.0% (p less than 0.001). There was no effect of warfarin on bone calcium content or dry weight in the absence of 1,25-(OH)2D3 treatment. These observations indicate that a component of the steroidal hormone action of 1,25-(OH)2D3 on bone may be mediated by increased synthesis of a vitamin K-dependent protein. The action of this vitamin K-dependent protein would oppose net calcium loss in the tibial metaphysis of 1,25-(OH)2D3-treated rats. This vitamin K-dependent protein may be the bone Gla protein, the only bone specific protein whose synthesis is known to be increased by 1,25-(OH)2D3.     

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction

A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by warfarin of prothrombin synthesis and of lipid-saccharide synthesis in rat liver

In vivo and in vitro studies using [³H]glucosamine incorporation into prothrombin and into glycolipids were conducted in rat liver to determine the role of lipid-saccharides in the biosynthesis of prothrombin.In vivo studies demonstrated that 10 mg warfarin/kg inhibited the incorporation of radiolabeled glucosamine into liver prothrombin and glycolipids. This inhibition was similar to the kinetics of inhibition of prothrombin synthesis in the liver.In vitro studies demonstrated a time-dependent increase in the incorporation of radiolabeled glucosamine into lipid-saccharides and prothrombin. This incorporation was inhibited 50% by 5 · 10^?4 M warfarin. Warfarin also inhibited the incorporation of radiolabeled glucosamine into glycolipids in a dose-related manner.In all studies, vitamin K-1 reversed the inhibition of glucosamine incorporation into glycolipids and into prothrombin.     

miR-200s Contribute to Interleukin-6 (IL-6)-induced Insulin Resistance in Hepatocytes

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 μg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.     

Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.

During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.     

Antimetabolic activity of L-ascorbic acid in human and animal tumors

1. The effect of high concentrations of L-ascorbic acid on the growth of some human and animal transformed and non-transformed cell lines has been investigated. Directly implemented into culture of transformed cell lines it decreased [3H]thymidine, [3H]uridine and [3H]leucine incorporation into cells. Vitamin C inhibited DNA synthesis by transformed cells 3-4 times more efficiently than by normal cells. 2. In vivo treatment of athymic nude mice bearing human mammary carcinoma with 500 mg/kg L-ascorbic acid for the first 15 days markedly inhibited the growth of tumor cells. 3. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in mammary carcinoma cells treated with vitamin C. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase K assay. Removal of vitamin C caused an immediate onset of spontaneous repair of single or double stranded DNA breaks. If, however, vitamin was reintroduced into cell culture, this spontaneous repair was reversed. 4. Our results indicate an antimetabolic activity of L-ascorbic acid in human and animal transformed cells, probably due to lethal damages in DNA.     

B16 tumor cells contain a warfarin sensitive vitamin K1 2,3 epoxide reductase

Using an adapted assay that requires an enzyme aliquot that forms only 5 pmoles vitamin K, we were able to demonstrate vitamin K1 2,3 epoxide reductase activity in cultured B16 mouse melanoma cells. The enzyme uses dithiothreitol, but not NADH as a reducing cofactor and is sensitive to inhibition by warfarin (2% residual activity at 10 micrograms/ml warfarin). Incubation of B16 cells in culture with 30 micrograms/ml warfarin leads to an 45% residual reductase as compared to normally cultured B16 cells. Combined with the reported presence of vitamin K dependent carboxylase in B16 cells and the cytotoxicity of warfarin towards B16 cells this suggests an active vitamin K cycle in these melanoma cells that may be essential for survival.     

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression

A simple method was devised for the maintenance of the chorioallantoic membrane (CAM) of chick embryos in organ culture. Explants of CAM survived for up to 5 days in this system and retained the characteristic three-layered morphology (ectoderm, mesoderm, and endoderm). Induction of the CAM calcium-binding protein (CaBP) by effectors of calcium metabolism was studied in these organ cultures. Vitamin K was found to elicit a seven- to eightfold increase in CaBP, whereas no increase in CaBP activity occurred on supplementation with vitamin A, parathyroid hormone, an analogue of vitamin D, vitamin D and its hydroxylated metabolites, or with elevated calcium levels. The vitamin K-mediated induction of CaBP was dose-dependent, inhibited by the vitamin K antagonists warfarin and dicoumarol, selective for vitamin K5, and maximal at the developmental stage (13-15 days of incubation) corresponding to the onset of calcium transport by the CAM in vivo. CaBP levels increased after 60-70 h in cultures of 13-15 day CAM supplemented with vitamin K and reached maximal levels around 80-90 h of culture. The CAM ectoderm underwent extensive proliferation and often assumed a villuslike morphology in the vitamin K cultures.     

Myelin Galactolipid Synthesis in Different Strains of Mice

Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17-21. However, the amount of myelin in the brains of F1 (B6 X D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelinogenesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14-, 17-, and 21-day-old D2, B6, and F1 mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenylyl sulfate:galactosylceramide 3'-sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.     

Transfection of glucosylceramide synthase antisense inhibits mouse melanoma formation

MEB4 murine melanoma cells synthesize G(M3) as the major ganglioside. Inhibition of G(M3) synthesis by a specific glucosylceramide synthase inhibitor resulted in reduced tumorigenicity and metastatic potential of these cells. We used a molecular approach--antisense transfection targeting the glucosylceramide synthase gene--to regulate glycosphingolipid synthesis by MEB4 cells and examine the influence on tumor formation. Antisense transfection inhibited the synthesis of the direct product of glucosylceramide synthase, glucosylceramide, and consequently G(M3) ganglioside, by MEB4 cells, reducing the concentration of G(M3) in the transfectants by up to 58%. Although neither morphology nor proliferation kinetics of the cultured cells was affected, the inhibition of glycosphingolipid synthesis and reduction of total ganglioside content caused a striking reduction in melanoma formation in mice. Only 1/60 (2%) of mice injected ID with 10(4) antisense-transfected MA173 cells formed a tumor, compared to 31/60 (52%) of mice receiving MEB4 cells and 7/15 (47%) of mice receiving the MS2 sense-transfected cells (p < 0.001 and p = 0.005, respectively). These findings demonstrate that stable transfection of glucosylceramide synthase antisense reduces cellular glycosphingolipid levels and reduces tumorigenicity, providing further experimental support for an enhancing role of gangliosides in tumor formation.     

Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus

Bacteroides melaninogenicus requires vitamin K for normal growth. Cells incubated in a vitamin K-free medium form defective cell envelopes. Studies with vitamin K-grown "K(+)" and vitamin K-depleted "K(-)" cells showed that [(14)C]choline and [(14)C]glycerol were not taken up, but several amino acids and acetate were incorporated to the same degree by both types of cultures. However, K(-) cells incorporated succinate to a greater degree than did K(+) cultures. The relative incorporation of succinate into ceramide phosphorylethanolamine and ceramide phosphorylglycerol was depressed compared with incorporation into phosphatidylethanolamine in K(-) cultures. B. melaninogenicus can be grown in serial subculture in the absence of vitamin K in the presence of 2.5 mg/ml of succinate. Under these conditions the relative incorporation of [2,3-(14)C]succinate and (32)P into ceramide phosphorylethanolamine and ceramide phosphorylglycerol is markedly depressed. Stimulation of phosphosphingolipid synthesis by vitamin K was shown by comparing the uptake of (32)P and lipid phosphorus levels of a succinate-grown, vitamin K-depleted culture supplemented with (32)P plus 0.1 micro g/ml vitamin K(1) with a similar culture supplemented with (32)P only. The phosphosphingolipids from the vitamin K-supplemented cells incorporated greater amounts of (32)P and had higher levels of phosphorus than did the ceramide phosphorylethanolamine and ceramide phosphorylglycerol of the culture without added vitamin K. It was further shown that vitamin K added to a vitamin K-depleted culture stimulated synthesis of ceramide phosphorylethanolamine and ceramide phosphorylglycerol 38 min and 60 min, respectively, following the addition of the vitamin; incorporation of (32)P into other phospholipids was unaffected.