Inhibition of glycerol-3-phosphate acyltransferase by analogs of glycerol-3-phosphate.

Analogs of glycerol-3-phosphate were tested as substrates or inhibitors of the glycerol-3-phosphate acyltransferases of mitochondria and microsomes. (rac)-3,4-Dihydroxybutyl-1-phosphonate, (rac)-glyceraldehyde 3-phosphate, (rac)-3-hydroxy-4-oxobutyl-1-phosphonate, (1S,3S)-1,3,4-trihydroxybutyl-1-phosphonate, and (1R,3S)-1,3,4 trihydroxybutyl-1-phosphonate were competitive inhibitors of both mitochondrial and microsomal sn-glycerol-3-phosphate acyltransferase activity. An isosteric analog of dihydroxyacetone phosphate, 4-hydroxy-3-oxobutyl-1-phosphonate, was a much stronger competitive inhibitor of the microsomal than the mitochondrial enzyme. Phenethyl alcohol was a noncompetitive inhibitor of both the microsomal and the mitochondrial acyltransferases. The product of the mitochondrial acyltransferase reaction with (rac)-3,4-dihydroxybutyl-1- phosphonate was almost exclusively (rac)-4-palmitoyloxy-3-hydroxybutyl-1-phosphonate. The microsomal acylation reaction generated both the monoacyl product and (S)-3,4-dipalmitoyloxybutyl-1-phosphonate. The apparent Km for (S)-3,4-dihydroxybutyl-1-phosphonate was 2.50 and 1.38 mM for the mitochondrial and microsomal enzymes, respectively.     

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.     

Properties of 3-hydroxypropionaldehyde 3-phosphate.

Several modifications to the synthesis of the diethyl acetal of 3-hydroxypropionaldehyde-3-P (HPAP) are described. HPAP is liberated from its acetal by treatment with Dowex 50-H⁺ at 40 °C for 4 min, and longer time or higher temperature lower yields. Breakdown of the dianion of HPAP (pK 6.7) is self-catalyzed, with the phosphate acting as a general base to remove a proton from carbon 2 and allow elimination of phosphate to give acrolein. Monoanion breakdown is at least 400-fold slower. At 25 °C the dianion breaks down with k = 0.025 min^?1, and the activation energy for the process is 24 kcal/mol. Buffers have little effect on breakdown of HPAP, except for those containing primary or secondary amines. Thus morpholine enhances breakdown by forming a Schiff's base with a positively charged nitrogen, and Tris inhibits breakdown by forming one with an uncharged nitrogen. The aldehyde group of HPAP is 60% hydrated in water.     

Enzymatic measurement of sphingosine 1-phosphate.

Sphingosine 1-phosphate (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including sphingosine. Extracted SPP is efficiently converted to sphingosine by alkaline phosphatase treatment. Sphingosine thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant sphingosine kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/10(6) cells. Concurrent measurements of sphingosine levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC12 cells after treatment with exogenous sphingosine or with nerve growth factor which stimulates sphingosine kinase activity.     

Sturgeon glyceraldehyde-3-phosphate dehydrogenase.

The formation of binary complexes between sturgeon apoglyceralddhyde-3-phosphate dehydrogenase, coenzymes (NAD+ and NADH) and substrates (phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate) has been studied spectrophotometrically and spectrofluorometrica-ly. Coenzyme binding to the apoenzyme can be characterized by several distinct spectroscopic properties: (a) the low intensity absorption band centered at 360 nm which is specific of NAD+ binding (Racker band); (b) the quenching of the enzyme fluorescence upon coenzyme binding; (c) the quenching of the fluorescence of the dihydronicotinamide moiety of the reduced coenzyme (NADH); (D) the hypochromicity and the red shift of the absorption band of NADH centered at 338 nm; (e) the coenzyme-induced difference spectra in the enzyme absorbance region. The analysis of these spectroscopic properties shows that up to four molecules of coenzyme are bound per molecule of enzyme tetramer. In every case, each successively bound coenzyme molecule contributes identically to the total observed change. Two classes of binding sites are apparent at lower temperatures for NAD+ Binding. Similarly, the binding of NADH seems to involve two distinct classes of binding sites. The excitation fluorescence spectra of NADH in the binary complex shows a component centered at 260 nm as in aqueous solution. This is consistent with a "folded" conformation of the reduced coenzyme in the binary complex, contradictory to crystallographic results. Possible reasons for this discrepancy are discussed. Binding of phosphorylated substrates and orthophosphate induce similar difference spectra in the enzyme absorbance region. No anticooperativity is detectable in the binding of glyceraldehyde 3-phosphate. These results are discussed in light of recent crystallographic studies on glyceraldehyde-3-phosphate dehydrogenases.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

2-deoxy-glucose-6-phosphate utilization in the study of glucose-6-phosphate dehydrogenase mosaicism.

The electrophoretic difference between normal glucose-6-phosphate dehydrogenase (G6PD) and two common variants (G6PD A and G6PD A-) has made the G6PD enzyme system very useful for genetic studies and for investigation on the clonal origin of tumors. This approach has not been possible for another common variant, G6PD mediterranean, which has a normal electrophoretic pattern. The different utilization of 2-deoxy-glucose-6-phosphate (2dG6P), an analog of the normal substrate, by the normal enzyme and the Mediterranean variant, allows a convenient determination of the degree of mosaicism in mononuclear cells from heterozygotes.     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

Characterization of murine sphingosine-1-phosphate phosphohydrolase.

In the present study we have characterized mammalian sphingosine-1-phosphate phosphohydrolase (SPP1), an enzyme that specifically dephosphorylates sphingosine 1-phosphate (S1P) and which differs from previously described lipid phosphate phosphohydrolases. Based on sequence homology to murine SPP1, we cloned the human homolog. Transfection of human embryonic kidney 293 and Chinese hamster ovary cells with murine or human SPP1 resulted in marked increases in SPP1 activity in membrane fractions that were used to examine its enzymological properties. Unlike other known type 2 lipid phosphate phosphohydrolases (LPPs), but similar to the yeast orthologs, mammalian SPP1s are highly specific toward long chain sphingoid base phosphates and degrade S1P, dihydro-S1P, and phyto-S1P. SPP1 exhibited apparent Michaelis-Menten kinetics with S1P as substrate with an apparent K(m) of 38.5 microm and optimum activity at pH 7.5. Similar to other LPPs, SPP1 activity was also independent of any cation requirements, including Mg(2+), and was not inhibited by EDTA but was markedly inhibited by NaF and Zn(2+). However, SPP1 has some significantly different enzymological properties than the LPPs: the aliphatic cation propanolol, which is an effective inhibitor of type 1 phosphatidate phosphohydrolase activities and is only modestly effective as an inhibitor of LPPs, is a potent inhibitor of SPP1; the activity was partially sensitive to N-ethylmaleimide but not to the thioreactive compound iodoacetamide; and importantly, low concentrations of Triton X-100 and other non-ionic detergents were strongly inhibitory. Thus, in agreement with Cluster analysis which shows that outside of the consensus motif there is very little homology between SPP1s and the other type 2 lipid phosphohydrolases, SPP1s are significantly different and divergent from the mammalian LPPs.     

Compartmentation of glucose 6-phosphate in hepatocytes.

Rat hepatocytes were incubated with 14C-labelled hexoses, and the specific radioactivities of glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate were determined. (1) When suspensions of freshly isolated hepatocytes were incubated with [14C]glucose, the specific radioactivities of glucose 1-phosphate and fructose 6-phosphate were severalfold higher than that of glucose 6-phosphate. The ratios of the specific radioactivities decreased with time of incubation. These relationships were also found when incubations were carried out with primary cultures of rat hepatocytes or with crude homogenates of hepatocytes, but not with isolated nuclei. (2) When cells were incubated with [14C]fructose, the ratios of the specific radioactivities were higher than with [14C]glucose, and also decreased with time. (3) Paired incubations were carried out with a mixture of galactose and fructose, with one or other sugar being labelled with 14C. The specific radioactivity of glucose released into the medium was greater than that of glucose 6-phosphate when fructose was labelled, but not when galactose was labelled. Furthermore, glucose 6-phosphate and glucose in the medium differed with regard to the distribution of 14C between C-1 and C-6. These results are interpreted as evidence that glucose 6-phosphate in hepatocytes does not exist as a homogeneous pool, but that subcompartments exist which are associated with glucose phosphorylation, gluconeogenesis and glycogenolysis.