Effect of vitamin B3-active compounds on the content of free and combined gamma-aminobutyric acid and glutamic acid in the brain of mice

The bound and free GABA and glutamic acid content in the brain of F1 (CBA X C57B1/6) hybrid mice was investigated by the Eliott method. A tendency to a decrease of GABA and glutamate content in the brain with their practically constant bound/free ratio was observed 24 h after calcium-D-pantothenate injections (150 mumole/kg, 9 injections for 3 days). Calcium-D-homopantothenate injected in the same way caused a significant decrease in the GABA content, and a sharp drop of the bound/free GABA ratio. The effect is not associated with the influence of calcium ions in the composition of the injected compounds.     

Effect of various respiration substrates and growth marker proteins on oxygen consumption in rabbit heart mitochondria

Oxygen uptake by myocardium mitochondria of healthy, carcinomatous and new-born rabbits in the presence of different substrates was studied under the effect of immunoglobulin G and beta-globulin peculiar to the normal and malignant growth. It is stated that the growth marker proteins representing a subfraction of immunoglobulin G of tumour patients blood serum and beta-globulin of new-born rabbits blood serum in the presence of pyruvate, alpha-ketoglutarate and glutamate inhibit the oxygen uptake by healthy rabbits heart mitochondria. Studies conducted on mitochondria of new-born and carcinomatous rabbits show that the action of the proteins under study depends on the respiration substrate. In the presence of succinate the proteins under study activate the oxygen uptake and pyruvate, alpha-ketoglutarate and glutamate inhibit this process. A conclusion is drawn that the effect of proteins peculiar to the growth process on the biological oxidation depends both on the substrate and structural state of mitochondria.     

Changes of the quaternary structure of microvillus aminopeptidase in the membrane

Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300 000. Normal preparations contain three different subunits (subunit A, Mr 162 000; subunit B, Mr 123 000; subunit C, Mr 61 000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.     

Introduction of aliphatic amino and hydroxy groups to keto steroids using O-substituted hydroxylamines.

(Aminooxy)butyl- and -hexylamines and alcohols were synthesized by the Ing-Manske modification of the Gabriel synthesis. The aminooxy group of these heterobifunctional spacer reagents is a far more powerful nucleophile than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (alpha-effect). The aminooxy group reacts readily with keto groups while the amino or hydroxy (or other) group remains free for further reactions. These excellent heterobifunctional spacer reagents were used here to derivatize keto steroids in an alkaline alcoholic solution. Using the described, general, and easy one-step synthesis, aliphatic amino or hydroxy groups with a spacer arm have been introduced to testosterone, 6-ketoestradiol, and cortisol.     

The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.     

Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane

Clathrin in coated vesicles is linked to transmembrane receptors by adaptor protein complexes. The Golgi-associated adaptor complex HA1 is a tetramer, made up of beta', gamma, 47-kDa, and 20-kDa subunits, whereas the tetrameric plasma membrane adaptor, HA2, contains alpha, beta, 50-kDa, and 16-kDa subunits (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929). Here we report on the structural organization of adaptor subunits as revealed by proteolytic dissection. We show that the beta' and gamma subunits of HA1 are cleaved into 60-67-kDa "trunk" and 32-44-kDa "head" fragments. Interactions between adaptor subunits involve the trunk domains only. In overall organization of their domains, the Golgi and plasma membrane adaptors are very similar. The similarity encompasses also the location of phosphorylated serine residues in the alpha a, beta, beta', and gamma subunits, which are found in the head domains in all cases. In the alpha a and beta subunits they probably occur in the proline- and glycine-rich hinge region, which connects the head to the trunk. Identical adaptor fragments were obtained by controlled digestion of clathrin-coated vesicles. Under conditions that did not affect the integrity of the clathrin heavy chain, the adaptor head fragments were always quantitatively released from coated vesicles. The release of the bulk of the adaptors occurred concomitantly with the cleavage of their beta-type subunits (beta and beta') and under buffer conditions that prevent aggregation of adaptors. These observations taken together with the results of reconstitution experiments confirm and extend previous data (Ahle, S., and Ungewickell, E. (1989) J. Biol. Chem. 264, 20089-20093) which suggested that adaptors attach to clathrin through their beta-type (beta and beta') subunits. Moreover, high affinity interaction between adaptors and clathrin requires the participation of regions from both the head and trunk domains of the beta-type subunits.     

The primary structure of the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin

The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.     

Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus

Cephalosporin biosynthetic activity and extracellular protease production increased during growth of Streptomyces clavuligerus in defined medium, while the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) remained very low and stable. Cephalosporin biosynthesis (measured in resting cell systems) was initiated during early exponential growth in complex media, without appreciable change in the small ppGpp pool. Nutritional shift-down induced by withdrawal of Casamino acids caused a transient increase in ppGpp and a reduction of RNA accumulation. The increase in ppGpp was small in very young cultures, but increased as the culture aged. Twenty-seven spontaneous thiostrepton-resistant mutants were isolated and partially characterized. Most of them had a reduced ppGpp-forming ability and gave normal titres of cephalosporin. However, in complex medium, some mutants did not produce cephalosporins or extracellular protease, whereas others overproduced cephalosporins. The results indicate that, in S. clavuligerus, there is no obligatory relationship between the initiation of secondary metabolism and the stringent response.