A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.     

The formation of ceramide-1-phosphate during neutrophil phagocytosis and its role in liposome fusion

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.     

Hydrogen isotope effects in the cyclization of D-glucose 6-phosphate by myo-inositol-1-phosphate synthase

myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testis, Acer pseudoplatanus L. cell culture and Oryza sativa L. cell culture, converted D-[5-3H]glucose 6-phosphate to myo-[2-3H]inositol 1-phosphate at rates ranging from 0.21 to 0.48 that of unlabeled substrate. D-[3-3H]- and D-[4-3H]glucose 6-phosphate were converted at approximately the same rate as that of unlabeled substrate. In the case of testis enzyme, storage as a frozen solution further lowered the rate with D-[5-3H]glucose 6-phosphate as substrate. When the reaction was run in [3H]water, no 3H appeared in myo-inositol 1-phosphate but a small amount was recovered in substrate isolated from the final reaction mixture. These data support the involvement of carbon 5 of D-glucose 6-phosphate in the mechanism proposed for this conversion.     

Identification of a novel growth factor-like lipid, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) that is present in commercial sphingolipid preparations

Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and 13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 microM was only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065-C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.     

Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase

We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. The ysr2Delta deletion mutant of S. cerevisiae accumulated DHS-1-P compared with its wild type strain upon labeling with D-erythro-[4, 5-3H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing the ysr2Delta deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling with D-erythro-[3H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.     

Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene

We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene encoding this enzyme. Pure enzyme was obtained by 64-fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleotide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catalyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a "ping-pong" type of mechanism. The Km values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.     

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.     

Polyol metabolism by a caries-conducive Streptococcus: purification and properties of a nicotinamide adenine dinucleotide-dependent mannitol-1-phosphate dehydrogenase

The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii) diethyl-aminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at -80 degrees C in the presence of beta-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenedia-minetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NAD-dependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)-dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate and NAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K(m) values for mannitol-1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K(m) values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine 5'-triphosphate and fructose-6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.     

Glycerol-3-phosphate acyltransferase in plants

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the transfer of an acyl group from an acyl donor to the sn-1 position of glycerol 3-phosphate. The plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm, respectively. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor. cDNAs for GPAT of chloroplasts have been cloned from several plants, and the gene for the enzyme has been cloned from Arabidopsis thaliana. The amino acid sequences deduced from the nucleotide sequences of cDNAs indicate that the product of translation is a precursor of about 460 amino acid residues, which consists of a leader sequence of about 70 amino acid residues and a mature protein of about 400 residues, with a molecular mass of about 42 kDa. Genetic engineering of the unsaturation of fatty acids has been achieved by manipulation of the cDNA for the GPAT found in chloroplasts and has allowed modification of the ability of tobacco to tolerate chilling temperatures.     

Lectin-like characteristics of recombinant human interleukin-1beta recognizing glycans of the glycosylphosphatidylinositol anchor

We found that 35S-labeled recombinant human interleukin-1beta (rhIL-1beta) binds phosphatidylinositol-specific phospholipase C-treated human placental alkaline phosphatase, phosphatidylinositol-specific phospholipase C-treated trypanosome surface variant glycoproteins, and urinary uromodulin immobilized on plates or immobilized on CNBr-activated Sepharose 4B. The interaction between rhIL-1beta and these glycoproteins was lectin-like, since it was inhibited in the presence of specific saccharides, i.e. mannose 6-phosphate or synthetic Ac-NH.CH2.CH2. PO4--->6Manalpha1-->(+/-2Manalpha1-->+/-6Manalpha1-->) propyl at about 1 microM. On the other hand, a wide variety of compounds including biantennary sugar chains derived from these glycoproteins as well as ethanolamine phosphate, inositol phosphate, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate did not show any inhibitory effect at concentrations up to 1 mM. These results indicate that rhIL-1beta interacts with these glycoproteins via the mannose 6-phosphate diester of glycans on the glycosylphosphatidylinositol (GPI) anchor. Furthermore, when monolayers of polarized Madin-Darby canine kidney cells on polycarbonate filter membranes were incubated with 35S-rhIL-1beta in either the apical or basolateral chamber, 35S-interleukin-1beta was found to bind specifically to the apical membranes with a Ka value of 4.6 x 10(7) M-1, and the specific interaction was inhibited by 1 microM mannose 6-phosphate. Since the mannose 6-phosphate diester moiety exists only in the GPI glycans on plasma membranes, it was evident that interleukin-1beta can directly interact with the mannose 6-phosphate diester component of the intact glycan of GPI anchors on plasma membranes.     

Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.     

Monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

The acyl-CoA:acyl-sn-glycerol 3-phosphate acyltransferases located in the microsomal fraction of lactating bovine mammary tissue show a preference for palmityl-CoA particularly above the apparent Km values of the acyl acceptors. Using saturating levels of monopalmityl-sn-glycerol 3-phosphate, the order of acylation was palmityl- greater than myristyl- greater than oleyl- greater than stearyl- greater than linoleyl-CoA. Apparent Km values for monopalmityl- and mono-oleyl-sn-glycerol 3-phosphate with palmityl-CoA as donor were 16 and 13muM, respectively while the Km values for palmityl-CoA with these two acyl acceptors were 5 and 5.2muM, RESPECTIVELY. The apparent Vmax values for the palmityl acceptor and donor were 25 and 30 nmol/min/mg protein. Phosphatidic acid was the principal product. The inclusion of magnesium in the assay depressed activity while the addition of ethylenediaminetetraacetate doubled the rate of acylation.     

Mannitol 1-phosphate mediates an inhibitory effect of mannitol on the activity and the translocation of glucokinase in isolated rat hepatocytes

When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.     

An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva

Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2.     

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.     

Carbocyclic analogues of D-ribose-5-phosphate: synthesis and behavior with 5-phosphoribosyl alpha-1-pyrophosphate synthetases

The synthesis of cyclopentyl and cyclopentenyl analogues of the alpha-anomer of D-ribose-5-phosphate from D-ribonolactone and D-ribose is described. These analogues, which have the same absolute configuration as D-ribose-5-phosphate, were incubated with PRPP synthetases in an attempt to prepare the corresponding carbocyclic PRPP analogues. The carbocyclic ribose-5-phosphate analogues were found to be inhibitors, rather than substrates, for 5-phosphoribosyl alpha-1-pyrophosphate synthetases of both bacterial and human origin. The inhibitory behaviour of the analogues is described.     

Phosphorylated cis-4-methylsphingosine mimics the mitogenic effect of sphingosine-1-phosphate in Swiss 3T3 fibroblasts

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155-167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analogue cis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative of cis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitor L-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative of cis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.     

Cryoinactivation and conformational drift of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

The cryoinactivation of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (GAPDH-rabbit) was studied. It was found that the inactivation of GAPDH-rabbit at 0 degrees C was much faster than that of GAPDH from yeasts, and showed obvious time and concentration dependence. The spectral properties, enzyme activity and behavior under pressure, of GAPDH-rabbit treated either by cryoinactivation, or pressure-induced dissociation and reassociation, were very similar. These results provided evidence to support the idea that cryoinactivation of oligomeric proteins, might take place through a cycle of dissociation-reassociation accompanied with the so-called conformational drift postulated by King and Weber (1986).