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.     

Immunoglobulin M possesses two binding sites for complement subcomponent C1q, and soluble 1:1 and 2:1 complexes are formed in solution at reduced ionic strength

Soluble complexes were formed between C1q, a subunit of the first component of human complement, and four different Waldenstr?m IgM proteins at reduced ionic strengths. The equilibria between these complexes and the free proteins were studied in the ultracentrifuge. Complex formation was found to be a very sensitive function of the salt concentration, and at physiological ionic strength complex formation was negligible. The complexes were cross-linked with a water-soluble carbodiimide and separated by sucrose gradient centrifugation. Both 22 S 1:1 and 26 S 2:1 C1q X IgM complexes were formed; stoichiometry was established by cross-linking 125I-C1q with 131I-IgM and determining the ratios of the specific activities of the gradient-purified materials. The association process was studied as a function of protein concentration and was analyzed by Scatchard and Hill plots to yield stoichiometry, association constant, and degree of cooperativity. The results indicated that IgM has two identical and independent binding sites for C1q. The intrinsic association constant was found to vary between 10(6) M-1 at 0.084 M ionic strength to 10(4) M-1 at physiological ionic strength; the slope of the log-log plot gave a value of -6.0. The cross-linked complexes were examined by electron microscopy, and the C1q appeared to be attached to the IgM through the C1q heads, implying that the biologically significant binding sites were involved in this interaction. For the 2:1 complexes, the two C1q appeared to attach to opposite surfaces of the IgM, suggesting the presence of a pseudo-2-fold axis lying in the plane of the IgM disk.     

Mammalian fertilization as seen with the scanning electron microscope

For several years we have been looking at mammalian gametes and their interactions with the scanning electron microscope (SEM). Examining the images produced by the SEM has given us a three-dimensional view of sperm, eggs, and egg investments. We are particularly impressed with the structural variation among gametes of different mammalian species. In this short report we examine the structure of mammalian spermatozoa, eggs, zonae pellucidae, and cumuli. Our observations and those of others have led us to believe that variation in gamete structure and function may have evolved as a mechanism for reproductive isolation of mammalian species.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

A note on the production and use of antisera for the detection of part-digested sticklebacks in predator stomach-content samples

Preliminary experiments indicate that serological techniques provide a useful method of identifying specific prey antigens from the stomach contents of fish predators. Problems with cross-reacting antigens from different prey species can be reduced by absorbing anti-sera with tissue extracts of the cross-reacting species. Very small volumes of part-digested prey tissue are sufficient for identification of the prey species when using agarose diffusion plates (Ouchterlony tests).     

Characterization of Schistosoma mansoni antigen-reactive T cell clones that form granulomas in vitro

Granuloma formation and modulation in schistosomiasis are a consequence of discrete subpopulations of T lymphocytes and the mediators they produce. In the present study, T cell clones reactive to soluble egg antigen (SEA) were developed to analyze the roles of T cells in Schistosoma mansoni egg-induced granuloma formation. In an in vitro granuloma assay, 1 X 10(5) T cells specifically augmented the response of 2 X 10(6) normal spleen cells to SEA-coupled but not purified protein derivative-coupled polyacrylamide beads. In vitro granulomatous responses by individual clones were correlated with their capacity to mediate local delayed-type hypersensitivity reactions in footpad swelling assays. Phenotypic analysis of the seven clones characterized in the present study demonstrated that they were L3T4+, Ly-2.2-. An analysis of supernatants of T cells pulsed with concanavalin A or SEA + antigen-presenting cells was also undertaken in an attempt to correlate in vitro granuloma formation with lymphokine production. Stimulated T cells (but not unstimulated T cells) produced interleukin 2, macrophage activating factor, migration inhibitory factor, and eosinophil stimulation promoter in response to both mitogenic and antigenic stimuli. The results suggest that individual clones of T cells are capable of producing a variety of mediators that influence their ability to activate and to recruit cells into granuloma formation. The model may be useful in the analysis of specific antigens and regulatory interactions and their contribution to granuloma formation.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.     

Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus.

The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. Relatively few differences in polypeptide composition exist when microaerobic cells are compared with anaerobic cells, but these changes correlate with a change from the mycelial to the yeast morphology. We hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis.