Inhibition by cycloserine of mitochondrial and cytosolic aspartate aminotransferase in isolated rat hepatocytes.

Isolated hepatocytes were incubated with L-cycloserine and then treated with digitonin so that mitochondrial and cytosolic fractions were obtained in 5 s. Mitochondrial and total cellular aspartate aminotransferases (EC 2.6.1.1) were inactivated in parallel. The enzyme was also inhibited in isolated mitochondria incubated with L-cycloserine. These results, in contrast with previous reports, indicate that cycloserine reacts equally with mitochondrial and cytosolic aspartate aminotransferases.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

l-2-Oxalylamino-3-aminopropionic acid,an isomer of Lathyrus sativus neurotoxin

The first chemical synthesis of l-2-oxalylamino-3-aminopropionic acid, an isomer of the Lathyrus sativus neurotoxin, is described. Studies on its biological properties are reported. Experiments with l-3-[¹⁴C-oxalyl]amino-2- aminopropionic acid show that the amount of 2-oxalylamino isomer detectable in seed extracts can be accounted for by rearrangement which occurs during isolation.     

Formation of glycine and aminoacetone from l-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from l-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM l-threonine, but exogenous NAD⁺ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of l-threonine, glycine being the major product at low l-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20 000 × g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous tow-carbon compound detected when intact mitochondria catabolized l-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from l-threonine in rat liver mitochondria occured primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Simultaneous determination of d- and l-thyroxine in human serum by liquid chromatography with electrochemical detection

A method for the determination of d- and l-thyroxine in human serum is described. The method involves extraction of thyroxine from serum and the separation of thyroxine enantiomers on a reversed-phase, high-performance liquid chromatographic column by use of a chiral eluent containing l-proline and cupric sulfate. Satisfactory resolution of the enantiomers of thyroxine, triiodothyronine, and reverse triiodothyronine can be achieved in 12 min and, employing amperometric detection to monitor the separation, the detection limit for serum thyroxine is in the range of 1–3 ng per injected sample.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

Rupture of rat liver lysosomes mediated by l-amino acid esters

Treatment of rat liver lysosome suspensions with 0.5–20 mM α-l-amino acid esters results in a progressive loss of latency of lysosomal enzyme activity. The increase in available acid phosphatase activity in lysosomal suspensions is correlated with the decrease of turbidity of these suspensions. Ester mediated turbidity decrease is dependent upon ester concentration, and the pH and ionic strength of the suspending medium. d-Stereoisomers of amino acid esters do not exhibit comparable capacity to damage lysosomes.α-l-Amino acid esters were found to be substrates for neutral lysosomal esterase and transpeptidase activity. The d-stereoisomers are degraded at much lower rates. These data support the hypothesis that ester dependent lysosomal rupture is mediated by the specific interaction of the ester with a structural or functional lysosomal protein.     

Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase

There is doubt about the l-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of l-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. l-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing l-threonine without and with CoA and oxaloacetate; l-[U-¹⁴C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from l-threonine to 10–14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised l-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from l-threonine by l-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l-3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K_i is 100 μm. It was shown that l-3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l-3,4-[¹⁴C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l-prolyl-tRNA. The addition of l-3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.     

Inhibition of 3-phosphoglycerate dehydrogenase by compounds other than l-serine

3-Phosphoglycerate dehydrogenase from etiolated pea epicotyls was not affected during in vitro assay by a range of hexose phosphates, amino group precursors and nucleotides at 1 mM but was significantly inhibited by 1 mM ATP and GTP. ADP and GDP gave slight inhibition at this concentration. NADH caused almost total inhibition at 0.45 mM.     

CNDO/2 calculations of the relative stability of poly (l-proline I) and poly (l-proline II)

The CNDO/2 method using the tight binding approximation for polymers was applied to poly(l-proline I) and poly(l-proline II). The calculations were also carried out for poly(l-alanines) and model molecules which have the same backbone geometrics as those of poly(l-prolines). The results obtained show that both forms of poly(l-proline I) and poly(l-proline II) have nearly the same energy in agreement with experimental results. From the analysis of the total energy, it was found that the intrasegment energy of poly(l-proline II) was lower than that of poly(l-proline I) while the intersegment energy of poly(l-proline I) was lower than that of poly(l-proline II). This result can be considered to correspond well with the experimental fact that poly(l-proline II) is more stable in good or polar solvents and poly(l-proline I) in poor or non-polar solvents. The analysis of the total energy of poly(l-proline) leads us to the conclusion that the α and β carbons play an important role in determining the relative stability between poly(l-proline I) and poly(l-proline II) and the γ carbon does hvae a marked effect on the electronic structures of the polymers in question. This conclusion was also confirmed by comparison of the electronic structures of poly(l-prolines) with those of poly(l-alanines) and model compounds concerned.     

The metabolism of l[3-3H]lactate by isolated hamster liver cells

The rate of tritium removal from l[3-³H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of ¹⁴C disappearance from l[U-¹⁴C]lactate, with the result that the ³H/¹⁴C ratio in residual lactate from l-[U-¹⁴C,3-³H]lactate decreases. However, addition of low concentrations (0.1 to 1.0 mM) of l-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from l-[3-³H]lactate and l-[U-¹⁴C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.     

Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

A partition chromatographic procedure utilizing a cationic exchange resin column in the Li⁺ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of R_e and R_dP^? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the R_e or R_dP^? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H⁺ form) released more than 80% of the KDO residues within 15 min. The heptose of the R_dP^? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the R_e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R_dP^? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.     

Active transport of l-sorbose and 2-deoxy-d-galactose in Saccharomyces fragilis

Sorbose and 2-deoxy-d-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars.The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-d-galactose and chemiosmotic coupling in the case of sorbose and 2-deoxy-d-galactose. The difference between the two mechanisms are discussed.Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport.The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Synthesis of 4,5-dideoxy-4-C-[(R,S)-phenylphosphinyl]-d-ribo- and l-lyxo-furanose and their 1,2,3-triacetates

2,3-O-Isopropylidene-d-ribose diethyl dithioacetal, prepared from d-ribose, was converted in three steps into the corresponding dimethyl acetal, which was monotosylated at O-5, and the ester oxidized at C-4 with pyridinium chlorochromate; addition of methyl phenylphosphinate to the resulting pentos-4-ulose derivative then provided (4R,S)-4,5-anhydro-2,3-O-isopropylidene-4-C-[(R,S)-(methoxy)phenylphosphinyl]-d-erythro-pentose dimethyl acetal. Hydrogenation of this compound in the presence of Raney Ni, followed by reduction with SDMA, hydrolysis, and acetylation, yielded the title compounds (seven kinds), the structures of which were established on the basis of their 400-MHz, ¹H-n.m.r. and mass spectra. A general dependence of the ²J_PH and ³J_PH values on the OPCH and PCCH dihedral angles provided an effective method for the assignment of the configurations and conformations of these 4-deoxy-4-phosphinyl-pentofuranoses.     

A synthesis of l-ristosamine and a derivative of its C-4 epimer

A synthesis of l-ristosamine from l-rhamnal is described, involving the sequence of reactions: methoxymercuration, tosylation, azide displacement, and reduction, which gave methyl α-l-ristosaminide (10). Acid hydrolysis then afforded l-ristosamine hydrochloride. Trifluoroacetylation of the hydrochloride of 10 followed by saponification and oxidation with ruthenium tetraoxide gave methyl 2,3,6-tri-deoxy-3-trifluoroacetamido-α-l-erythro-hexopyranosid-4-ulose (17). Borohydride reduction of 17 gave a separable, 1:1 mixture of methyl 2,3,6-trideoxy-3-trifluoroacetamido-α-l-ribo- and α-l-xylo-hexopyranoside.     

Simultaneous determination of d- and l-thyroxine in human serum by liquid chromatography with electrochemical detection

A method for the determination of d- and l-thyroxine in human serum is described. The method involves extraction of thyroxine from serum and the separation of thyroxine enantiomers on a reversed-phase, high-performance liquid chromatographic column by use of a chiral eluent containing l-proline and cupric sulfate. Satisfactory resolution of the enantiomers of thyroxine, triiodothyronine, and reverse triiodothyronine can be achieved in 12 min and, employing amperometric detection to monitor the separation, the detection limit for serum thyroxine is in the range of 1–3 ng per injected sample.