Hoppe-Seyler's Zeitschrift fur physiologische Chemie最新文献

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).

Poly(ADP-ribosylation) was demonstrated in the intestinal parasite Ascaris suum, especially in the reproductive tissues. The activity of the ADP-ribosyltransferase was found to depend on divalent cations and to be stimulated by deoxyribonuclease I about 5-fold. The reaction rate was optimal at a temperature of 30 degrees C and at pH about 8.4. The apparent Km value for NAD was estimated to be 0.2mM. The enzyme activity was effectively inhibited by nicotinamide (Ki = 65 microM) benzamide (6 microM), 3-aminobenzamide (10 microM), theophylline (35 microM) and thymidine (50 microM). The type of inhibition by these compounds was found to be competitive with respect to NAD.

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.

The formation and fate of 3-dehydroecdysteroids were studied in vitro in tissue extracts from Pieris brassicae (Lepidoptera). Three cytosolic enzymes are involved: an ecdysone oxidase converts 3 beta-hydroxyecdysteroids into 3-dehydroecdysteroids, and two different reductases, requiring NADPH as cofactor, transform 3-dehydroecdysteroids into the 3 alpha-hydroxy- or the 3 beta-hydroxy forms, respectively. 3 alpha-Hydroxyecdysteroid formation is only detected in the gut; all tissues contain the two other activities. It appears that conversion of 3 beta- to 3 alpha-hydroxyecdysteroids proceeds through 3-dehydro compounds. The biological significance of these reactions is discussed.

The trypsin-kallikrein inhibitor aprotinin was modified with lactose. The influence of reactant concentrations, temperature, reaction time and sodium borohydride on the carbohydrate residue content and the inhibiting activity of glycated aprotinin were studied. Glycation of aprotinin neither shifts the pH optimum of the inhibitor-trypsin association reaction nor does it alter the apparent dissociation constant Ki of the complex measured at pH optimum. Glycation by lactose stabilizes aprotinin against denaturation by increased temperature. The distribution of native and modified aprotinin in rat organs after endocardiac injection was studied. Fixation of glycated aprotinin increases 2.5- to 3-fold in liver and decreases 2-fold in kidneys during the observation time (5 min-2 h) compared to native aprotinin.

The oxidation of human alpha 1-proteinase inhibitor results in the conversion of this protein into a form which cannot protect lung elastin from degradation by elastolytic proteinases. Data indicate that this is primarily because of the lowering of the association rate between the modified inhibitor and neutrophil elastase, as well as in a change in Ki from near 10(-14) to near 10(-10)M. This is consistent with the hypothesis that oxidation of alpha 1-proteinase inhibitor in the lung by cigarette smoke results in a lowering of the protection of this organ from elastolytic degradation.

The amino-acid sequences of the alpha- and beta-chains of gayal hemoglobin have been determined and compared with those of bovine and yak hemoglobins. The gayal alpha-chain differs from the alpha-chains of bovine by 3 amino-acid residues and from yak I alpha- and II alpha-hemoglobins by 4 and 2 residues, respectively. The gayal beta-chain differs from bovine beta A- and beta B-chains by 3 and 4 residues, respectively and from yak beta-chains by 2 residues.

Four low-molecular-mass polypeptides were isolated and purified from chromatophore membranes of Rhodopseudomonas sphaeroides blue-green mutant R-26.1 by a combination of gel filtration and ion-exchange chromatography in organic solvents. On dodecyl sulfate polyacrylamide gels, the purified polypeptides comigrate with bands LH-1, LH-2 and LH-3 known to be related to the antenna-pigment-protein complexes. The complete primary structures were elucidated by automated Edman degradation of the intact polypeptides and of overlapping C-terminal fragments obtained after chemical cleavage at tryptophan and methionine residues. The C-termini were verified by hydrazinolysis and, in one case where an overlapping C-terminal fragment could not be obtained, by digestion with carboxypeptidase A. The four polypeptides show a tripartite structure: i.e. a polar N-terminal region is separated from a polar C-terminal region by a segment of about 21 predominantly hydrophobic amino-acid residues. All hydrophobic segments contain a characteristic conservative histidine residue. The C-terminal region is reduced to only a few amino acids in the two polypeptides which together form band LH-3, i.e. LH-3A and LH-3B. Their extended N-terminal region is rich in charged residues and contains an additional conserved histidine residue close to the beginning of the hydrophobic segment. These properties place LH-3A and LH-3B into subgroup (beta-polypeptides: B 870-beta and B 850-beta, respectively). LH-1 and LH-2 appear to form another subgroup (alpha-polypeptides: B 870-alpha and B 850-alpha, respectively) as suggested during a search for conservative elements within their sequences (structural basis for classification). N-Terminal analyses carried out with intact antenna-pigment-protein complexes revealed the following: (i) LH-1 and LH-3 are associated with the B 870 complex in Rp. sphaeroides 24.1 (wild type), (ii) the same polypeptides are almost exclusively present in chromatophore membranes of Rp. sphaeroides R-26, a blue-green mutant which absorbs at 870 nm, (iii) LH-2 and LH-3B are the constituent polypeptides of the B 800-850 complex of Rp. sphaeroides 2.4.1 and of the spectrally altered B 850 complex isolated from the blue-green mutant R-26.1 which absorbs at 860 nm. This mutant contains LH-2 and LH-3B along with LH-1 and LH-3A and apparently is able to form both types of antenna complexes.(ABSTRACT TRUNCATED AT 400 WORDS)