Characterization of the S'-subsite specificity of V8 proteinase via acyl transfer to added nucleophiles

The S'-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P'1 position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.     

Evaluation of experimental combined toxicity by use of dose-frequency curves: comparison with theoretical additivity as well as independence

Dose-frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve-fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the phi 2-square goodness-of-fit test. The theoretical dose-frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose-response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

Die Variabilität vegetativer und generativer Flechtenstrukturen und ihre Bedeutung für die Systematik

Die Entstehung von Stützleisten in den Thalli von Cladonia chlorophaea und Cetraria islandica, sowie die Entwicklung sekundärer Soredien im Inneren der Podetien von Cladonia chlorophaea werden beschrieben. Gemeinsam mit anderen Entwicklungsvorgängen von Flechten zeigen die Beobachtungen eine sehr geringe Determination der Flechtengewebe. Die Struktur wird weitgehend durch die Funktion bestimmt, während gleichzeitig bestimmte ontogenetische Abläufe unveränderbar sind. Die Bedeutung dieser Erscheinungen für die Systematik wird diskutiert.     

Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.     

Stabilization by ATP and ADP of Escherichia coli dnaB protein activity

The effect of adenine ribonucleotides on the stability of Escherichia coli dnaB protein in cellular crude extracts was studied. Stabilization of dnaB protein by ATP or ADP, but not by AMP, was manifested in that (i) the activity and yield of wild type dnaB protein is enhanced in the presence of ATP, (ii) the dnaB protein of E. coli dnaB mutants, such as groPB and dnaB252/ColE1::dnaC+, which is inactive in a dnaB complementation assay, can be isolated in active form in the presence of ATP or aDP, (iii) ATP or ADP protect the dnaB protein of an E. coli dnaBts mutant from inactivation at 37 degrees C, and (iv) inactive groPB and dnaBts protein can be reactivated partially by ATP. Thus, the stabilizing effect of ATP and ADP can be exploited for the isolated of otherwise inactive or labile mutant dnaB proteins.     

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).