Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).     

Single-Cell Protein Production in Alkaline Mesquite Wood Extracts

A nine-member, mixed, cellulolytic, bacterial culture was used to evaluate the effects of sodium hydroxide normality, length and temperature of treatment, and the ratio of volume of alkali to mesquite wood on the suitability of alkali mesquite wood extractives as nutrients for bacterial growth. The presence or absence of air during the extraction process did not significantly affect results. The amount of lignin extracted and the total loss in weight of the wood during extraction were correlated to both alkali concentration and temperature. Neutralized extracts supported bacterial growth; growth was inversely related to the final salt concentration of the neutralized extract. Deionized extracts were superior to acid-neutralized extracts for the support of bacterial growth. The optimum conditions for extraction were 2.5 N NaOH at 30°C for 12 h. The study demonstrated that nutrients as well as growth inhibitory compounds are released from wood by alkali treatment. This study of alkali wood extracts and the previous study of washed alkali treated wood residues provide a data base for the optimization of alkali treatments of hard woods that are to be used as nutrients for the growth of cellulolytic cultures.     

Evaluation of factors affecting the membrane filter technique for testing drinking water.

The following studies were done in response to questions regarding the adoption and use of the membrane filter (MF) technique for testing drinking water for the total coliform indicator group. A comparison with the most-probable-number technique showed that MF procedures with m-Endo agar LES were somewhat superior to the most-probable-number methods in terms of numbers of coliform organims recovered. Medium preparation and storage studies indicated that rehydration of m-Endo agar LES should be done with boiling water for less than 15 min, that m-Endo agar LES should not be exposed to light for more than 4 to 6 h, and that m-Endo agar LES plates may be used for up to 4 weeks and broth verification media for up to 3 weeks under given storage conditions. MF culture colonies were commonly found which did not produce sheen as expected for coliforms and yet were verified as coliforms. The occurrence and morphology of these atypical colonies were studied. Parallel inoculation of both lauryl tryptose (LT) and brilliant green bile (BGB) broth was found to be a better colony verification approach than recommended LT preenrichment before transfer to BGB. Comparison of parallel verification results indicated very little justification for the use of LT medium in MF verification procedures. In the case of overgrown or confluent cultures, the best coliform recoveries resulted from swabbing the MF plate and directly inoculating BGB medium with the swab. The occurrence of overgrowth was defined and evidence was collected suggesting that overgrowth is a function of sample holding time. Evaluation of routine test data and bacterial population reductions as a function of time indicated that nonquantitative recovery of coliforms may not be significantly affected for at least a 72-h sample holding time.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Kininogen and Kinin in Experimental Spinal Cord Injury

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.     

Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin.

IgE-binding protein (epsilon BP) was originally identified by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain. The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains. The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage. In this study, human epsilon BP was first expressed in Escherichia coli, and the carboxyl-terminal domain (epsilon BP-C) was then generated by collagenase digestion of epsilon BP. By equilibrium dialysis, the association constants of epsilon BP and epsilon BP-C for lactose were found to be similar (6.0 +/- 0.70) x 10(4) M-1 and (4.7 +/- 0.27) x 10(4) M-1, respectively. Both polypeptides contain only one lactose-binding site/molecule. By an assay involving binding of 125I-labeled epsilon BP or epsilon BP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that epsilon BP-C retains the saccharide specificity of epsilon BP. Importantly, although unlabeled epsilon BP-C inhibited the binding of the radiolabeled epsilon BP to IgE, unlabeled epsilon BP caused increased binding to IgE, suggesting self-association among epsilon BP molecules. Oligomeric structures resulting from self-association of epsilon BP were confirmed by chemical cross-linking studies. Furthermore, epsilon BP possesses hemagglutination activity on rabbit erythrocytes, whereas epsilon BP-C lacks such activity. Based on these results, we propose a structural model for multivalency of epsilon BP: dimerization or oligomerization of epsilon BP occurs through intermolecular interaction involving the amino-terminal domain.