Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

Treatment of dialysis membranes for simultaneous dialysis and concentration

Dialysis membranes used for simultaneous dialysis-concentration required pretreatment to remove uv-absorbing compounds leached from the membranes and to reduce the absorption of protein to the membranes. This was accomplished with sodium carbonate and ethanol or with "sulfur-removal solutions." Protein determinations were made with a micro-Bradford protein reaction and with uv absorbance at 280 nm. Soluble membrane components contributed to aberrant uv spectra and altered the ratio of 280/260-nm absorbance. Simultaneous dialysis and concentration in the micro protein dialyzer-concentrator apparatus, combining aspects of thin-layer dialysis and ultrafiltration, resulted in rapid removal of salts from the protein solutions. Prior treatment of membranes reduced uncertainties in retentate recoveries, eliminated uv-absorbing components of membranes, and improved recoveries of protein.     

Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina

In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP-Y) can be accomplished by ethanol treatment of the native enzymes, or freeze-thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins.     

The essential role of L-glutamine in lymphocyte differentiation in vitro

The biochemistry of human B lymphocyte differentiation to plasma cells is incompletely understood. L-glutamine appears to be required for both lymphoblastic transformation and plasma cell formation in pokeweed-mitogen-stimulated human peripheral blood mononuclear cell cultures. Cells cultured with pokeweed mitogen in glutamine-deficient RPMI-1640 with 10% heat-inactivated and dialyzed fetal bovine serum were unable to incorporate 3H-thymidine or undergo morphologic lymphoblastic transformation assessed at 72 hours. However, 3H-thymidine incorporation could be maximally restored with as little as 0.08 mM L-glutamine or by using nondialyzed heat-inactivated fetal bovine serum, containing approximately. 1 mM L-glutamine. In subsequent cultures, using glutamine-deficient RPMI-1640 with 10% nondialyzed heat-inactivated fetal bovine serum, lymphoblastic transformation was equivalent with or without additional L-glutamine supplementation. However, only cultures with 2 mM L-glutamine supplementation underwent plasma cell differentiation as assessed by cytoplasmic staining with fluorescein-conjugated anti-immunoglobulin. When the kinetics of cellular immunoglobulin synthesis and secretion were analyzed by 3H- leucine incorporation into immunoglobulin, synthesis was 2-5 fold greater, and secretion 3-10-fold greater in cell cultures with 2 mM L-glutamine supplementation. By electron microscopy, only the glutamine-supplemented cells showed development of rough endoplasmic reticulum consistent with active immunoglobulin production. L-glutamine supplementation had no apparent effect on cell recovery, viability, % B cells, % T cells, % monocytes, or % helper and suppressor T cells. Thus, L-glutamine is essential for both lymphoblastic transformation and plasma cell differentiation. Future investigation of the selective nutritional requirements of cultured cells should yield further insights into the biochemical control of immune cell differentiation and function.     

Carbohydrate metabolism in transforming lymphocytes from the aged

There is an age-related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elderly suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.     

H-2-linked Ir gene control of T cell recognition of the Sm nuclear autoantigen and the aberrant response of autoimmune MRL/Mp-+/+ mice

We have examined T cell recognition of the nuclear autoantigen Sm. Rabbit Sm-primed cells from autoimmune MRL/Mp-+/+ (+/+) mice and from all normal strains tested were able to proliferate to rabbit Sm in vitro. In contrast, the reactivity of normal strains to Sm of murine origin was genetically restricted; only H-2f strains B10.M and A.CA, and H-2s strains B10.S and A.SW could recognize mouse Sm, suggesting that responsiveness to mouse Sm was under the control of H-2-linked Ir genes. Although five Iak-bearing normal strains (B10.A, B10.A(2R), B10.BR, A/Sn, and CBA) did not recognize mouse Sm, autoimmune +/+ (Iak) mice were responders. The responsiveness of the +/+ mice to Sm was probably not due to differences in their Iak region, compared with other strains, because the Iak region of normal strains and the autoimmune +/+ strain were indistinguishable by interstrain MLC, immune response gene product function, and recognition by anti-Iak mAb. Inhibition of Sm-induced proliferation by mAb demonstrated that T cells from autoimmune +/+ mice, responder normal strains, and nonresponder normal strains recognized rabbit and mouse Sm in the context of I region-encoded products. The T cell response to Sm antigen in normal mice is therefore Ia region restricted and, for the murine antigen, under Ir gene control. Autoimmune mice that spontaneously make anti-Sm antibodies (+/+) also perceive Sm in an Ia-restricted manner, but their responder status abrogates H-2-linked Ir gene control.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.     

Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors

Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.     

The potential of production of sulfated polysaccharides from Porphyridium

Summary The environmental conditions prevailing in Israel make marine algae an attractive crop for the production of valuable chemicals. A marine species of Porphyridium seems to fit this purpose.The unicellular red alga Porphyridium is encapsulated by a polysaccharide envelope that is present in the gel state. This polysaccharide is an acidic heteropolymer composed of sulfated sugars. It forms ionic bridges through divalent cations, thus reaching a very high molecular weight. The thickness of the polysaccharide capsule varies according to the phase of growth and the growth conditions. Its outer part dissolves in the growth medium, which becomes progressively more viscous. Sulfated polysaccharides form theramlly reversible gels similar to agar and carrageenan, which are usually extracted from marine macroalgae. These gels have been finding increasing use in commercial applications as gelling agents, thickeners, stabilizers, and emulsifiers.We have done experiments on the cultivation of a marine species of Porphyridium for the production of polysaccharides. This unicellular alga has an advantage over the macroalgae due to its relatively faster growth rate and the possibility to regulate its growth. The potential for production of the polysaccharide, both that dissolved in the external medium and that attached to the cell (including an intracellular fraction), and the effects of growth conditions on productivity were suudied in the laboratory. Porphyridium was also cultivated outdoors in seawater in 1-m² ponds and its growth potential investigated.