Sex difference in L-glutamine D-fructose-6-phosphate aminotransferase activity of mouse submandibular gland

L-Glutamine D-fructose-6-phosphate aminotransferase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino transferring), EC 5.3.1.19) activities in the three main salivary glands of male and female mice were measured. It was found that the activity in the submandibular gland was about 10 times more in females than in males, whereas the activities in the sublingual and parotid glands of males and females were similar. The activity in the submandibular gland of female mice was not affected appreciably by ovariectomy but it decreased to the level in males on injection of testosterone. The activity in males was not affected appreciably by injection of progesterone or 17β-estradiol, but it increased to the level in females after castration. The increased acitivity in castrated male mice was decreased again to the normal level by testosterone injection. Thus, this sex difference is caused by androgen, not by female hormones. On the basis of in vivo experiments using actinomycin D, it was suggested that testosterone produced an “enzyme inhibitor”, which suppressed the enzyme activity in the submandibular glands of androgen-rich animals.     

Effect of asparaginase on cell membranes of sensitive and resistant mouse lymphoma cells

High concentrations of Escherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containing Acinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells. Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolated and maintained in asparagine depleted or asparaginase containing medium. The E. coli asparaginase preparation inhibited protein and glycoprotein biosynthesis to comparable degrees. It did not have proteolytic or glycolytic activity. Escherichia coli asparaginase did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentrations of E. coli asparaginase have a specific effect on the Con A receptor in the sensitive line.     

Purification and properties of L-alanine: 5-ketooctanal aminotransferase from Conium maculatum

L-Alanine:5-ketooctanal aminotransferase has been isolated from leaves of Conium maculatum and purified 369-fold. The enzyme MW 56 200 catalyse     

Electrokinetic studies of the testosterone-aqueous d-glucose interface

Electro-osmosis and streaming-potential measurements were made across a testosterone-plug membrane, using water and aqueous solutions of d-glucose as permeants. The electrophoretic velocity of testosterone particles dispersed in these solutions was also measured, experiments being confined to the range where linear flux-force relationships hold. Phenomenological coefficients were evaluated by using these linear relations, and the results analyzed inthe light of the thermodynamics of irreversible processes. Saxen's relationship holds between electro-osmosis and streaming potential. Concentration dependence of the various phenomenological coefficients was also examined. Cross-phenomenological coefficients were found to decrease with increase in the concentration of d-glucose solutions. The results are explained on the basis of strong hydrogen-bonding between d-glucose and the surrounding water molecules. Such membrane parameters as pore size, average number of pores, and the membrane constant were evaluated. Electro-osmotic and electrophoretic data were used to estimate the zeta potential, in order to characterize the membrane-permeant interface. The dependence of the zeta potential on the concentration was also examined.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-γ-lactone : oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of FAD.     

Effects of temperature on L-leucine transport in toadfish liver in vivo

The effect of body temperature in the 4–30°C range on L-leucine uptake by toadfish liver in vivo was examined by means of a single-injection pulse technique. The ratio of [¹⁴C]leucine to [³H]mannitol or [³H]inulin in blood leaving the liver was measured as a function of time after hepatic portal vein injection. Recoveries of the two isotopes in liver and [¹⁴C]leucine incorporation into protein were determined.The Q₁₀ value for influx was 3.8, that for efflux 2.8. At all temperatures, the leucine influx was 8–10-times higher than its incorporation into protein. The directly energy-linked reactions appear to be the main site of increased temperature sensitivity at low temperatures.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Effect of L-leucine on p-aminohippurate accumulation by mammalian renal cortical slices

The effect of several amino acids (L-glutamate, L-phenylalanine, L-leucine, glycine, L-tryptophan, L-histidine, L-valine) on p-aminohippurate accumulation was evaluated in rat, mouse and rabbit kidneys. Only leucine was found to enhance p-aminohippurate accumulation in rat and mouse renal cortical slices but not in rabbit slices. Leucine had no effect on tetraethylammonium accumulation. In rat renal cortical slices, leucine increased the uptake and decreased the runout of p-aminohippurate, each effect contributing to the increase in p-aminohippurate accumulation. The apparent K_m of p-aminohippurate uptake was decreased by leucine with no change in the apparent V. Aminooxyacetate (an inhibitor of transamination of leucine) partially depressed the stimulating effect of leucine on p-aminohippurate accumulation, whereas α-ketoisocaproic acid (a metabolite of leucine formed by transamination) enhanced p-aminohippurate accumulation, suggesting that the metabolism of leucine in kidney slices may be necessary for the stimulating effect on p-aminohippurate transport.     

Synthesis of L-fucose 2-, 3-, and 4-Sulphates

Treatment of L-fucose with an excess of pyridine-sulphur trioxide gave an equilibrium mixture of mono-, di-,and tri-sulphates. L-Fucose was sulphated under optimal conditions for monosulphate formation, and the monoester fraction was isolated by chromatography on DEAE-cellulose. The isomeric L-fucose 2-, 3-, and 4-sulphates (1-3) were separated on a DEAE-cellulose column by elution with borate buffer. The structures of 1-3 were established by electrophoresis, colour tests, periodate oxidation, and, for the 2-isomer, by comparison with a specimen of 1 that had been definitively synthesised via methyl 3,4-O-isopropylidene-α-L-fucopyranoside (6) and methyl α-L-fucopyranoside 2-(barium sulphate) (5). The latter was rapidly hydrolysed in hot, dilute acetic acid to 1 and methyl α-L-fucopyranoside (4).     

Growth of a mutant of Escherichia coli K-12 on xylitol by recruiting enzymes for D-xylose and L1,2-propanedol metabolism

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.     

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.     

Immunochemical studies on L-rhamno-D-mannans of sporothrix schenckii and related fungi by use of rabbit and human antisera

Antisera were prepared in rabbits against the human pathogenic yeast Sporothrix schenckii (strain 1099.12) grown at two different temperatures (25° and 37°). Precipitation and inhibition data showed that the former serum had a specificity directed against α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-D-Man-(1→ determinants, whereas the latter had a broad specificity in which α-L-rhamnosyl or α-L-Rhap-(1→3)-D-Man-was the immunodominant structure. These results are consistent with data on the structures of the L-rhamno-D-mannans isolated from the organism grown at the two different temperatures. Human sera from patients with sporotrichosis were shown to have different specificities resembling the specificities developed in the rabbits. The rabbit antisera were also used to examine the cross-reactivity with L-rhamno-D-mannans from species of the genus Ceratocystis, which is reputed to include the ascigerous (perfect) state of S. schenckii. Polysaccharides from four species of Ceratocystis grown at 25° reacted with the antisera in a manner resembling that of the L-rhamno-D-mannan from S. schenckii grown at 37°. This is in accord with earlier data that showed that only S. schenckii, of the species studied, produces a polysaccharide with large amounts of α-L-Rhap-(1→2)-α-L-Rhap-(1→ side-chains when grown at 25°.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Electric field-induced orientation of l- and dl-phosphatidylcholine bilayers

Membrane orientation induced by an alternating electric field has been examined for the l-enantiomer and racemic dipalmitoylphosphatidylcholine (DPPC) bilayers. The orientation effect was measured by bending curvature of hairpin-like deformation of the multilamellar cylindrical tubes with varying field-strength, frequency and tube size. It has been observed that both l- and dl-DPPC tubes are similar in the profiles of field-strength dependence and frequency dependence on the curvature deformation, but different in the deformed curvatures. dl-DPPC tubes deform largely as compared with l-DPPC tubes. The square of the deformed curvature of dl-DPPC tubes is larger than that of l-DPPC by about 37% on average. The result indicates that the racemic membrane is responsive to the electric field as compared with the l-enantiomer membrane. This suggests that a hybrid arrangement of head groups of the racemic lipid leads an effective response of the membrane due to the head group orientation.     

Effect of asparaginase on cell membranes of sensitive and resistants mouse lymphoma cells

Summary High concentrations ofEscherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containingAcinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells.Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolate and maintained in asparagine depleted or asparaginase containing medium. TheE. coli asparaginase preparation inhibited protein and glycoprotein biosythesis to comparable degrees. It did not have proteolytic or glycolytic activity.Escherichia coli asparaginases did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentration ofE. coli asparaginase have a specific effect on the Con A receptor in the sensitive line. Results of the lecting binding studies were presented at the Federation meeting in Atlanta, GA, 1981. This work was supported by U.S. Public Health Service Grant CA20061, the Midwest Athletes Against Childhood Cancer Fund, and the Burroughs Wellcome Fund.     

Interaction properties of d-galactose-depleted guar galactomannan samples

The interaction of galactose-depleted guar galactomannan with agarose has been studied by optical rotation, and with xanthan using an Instron Materials Tester and a Rheometrics Mechanical Spectrometer. Samples were prepared by treatment of guar galactomannan with highly purified α-d-galactosidase. Modified guar galactomannans with a d-galactose content of 19–25%, in admixture with agarose, showed similar optical rotation changes on heating and cooling as did mixtures of agarose and carob galactomannan (23% d-galactose content). Modified samples with 13–16% d-galactose, in the presence of agarose, showed more pronounced optical rotation changes on heating and cooling, but samples with less than 13% d-galactose were only sparingly soluble even on autoclaving. The degree of interaction of galactose-depleted guar galactomannan samples with xanthan, as measured rheologically, increased as the d-galactose content decreased, paralleling the optical rotation changes with galactomannan/agarose mixtures. In the presence of xanthan, samples with a d-galactose content of 25% or less formed firm rubbery gels.