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

Liver regeneration after partial hepatectomy is characterized by an active phase of cell proliferation that is associated with a marked increase in thymidine kinase activity. Using non-denaturing gel electrophoresis and different substrate specificity, two isozymes could be detected. One was identified as the adult isozyme while the other was the fetal one. Both forms were present during liver regeneration. When the regenerative process was completed, the total enzymatic activity dropped to normal values and the fetal isozyme was displaced by the adult type.

The synthesis of two C-terminal peptides of bovine insulin B-chain are described. Thus, insulin fragments (B9-30) and ( B20 -30) were synthesized using nitrobenzoylglycyl -poly-(oxyethylene) as the soluble support. 4-Carboxy-2-nitrobenzyl ester of Boc-alanine was coupled to glycyl-poly(oxyethylene) and the syntheses were continued employing symmetrical anhydrides of Boc-amino acids. The protected peptides were cleaved from the support by photolysis and were purified on silica gel and Sephadex LH-20. All the protecting groups of a sample of the undecapeptide were removed with liquid HF and the unprotected peptide was purified on CM-cellulose. The synthesized peptides were gas chromatographically tested for the racemization of the individual amino acids. The results indicated that no residue was significantly racemized .

Cigarette smoke was found to be rather ineffective in inactivating alpha 1-proteinase inhibitor (alpha 1-PI) in aqueous solution, whereas a slow inactivation of alpha 1-PI by a dimethyl sulfoxide extract of whole cigarette smoke condensate was observed. However, this inactivation could only partially be prevented by antioxidants indicating that it is not, or at least not exclusively, due to oxidation. The bulk of inactive alpha 1-PI found in lung lavage fluids from smokers is thus probably generated through endogenous mechanisms and not through smoke components directly.

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.

A sialidase was solubilized with the aid of Triton X-100 from the insoluble material of a leucocyte homogenate. The enzyme was purified almost to homogeneity by chromatography on Sephadex G-75, equine submandibular gland mucin bound to Sepharose 4B and on Sephacryl S-200. The purification factor was 40 based on an increase of the specific enzyme activity from the Triton X-100 extract (pure enzyme: 40 mU/mg protein). Isolation of the active enzyme required the presence of a proteinase inhibitor. The sialidase is monomeric and has an average molecular mass of 48500 Da, a pH optimum of 4.6, hydrolyses preferably glycoprotein (fetuin) and sialyllactose, is activated by Ca2 and inhibited by N-acetyl-2,3-dehydro-2- deoxyneuraminic acid ( Neu5Ac2en ), Hg2 and N-(4-nitrophenyl) oxamic acid. The relatively stable enzyme shows only low activity with gangliosides and no activity with 4-O-acetylated sialic acid bound glycosidically.

Synthesis, degradation and leakage of lactate dehydrogenase and of total protein was measured using D-galactosamine-treated rat hepatocytes in monolayer culture. The kinetics of [3H]leucine incorporation into trichloroacetic acid-precipitable material and into isolated lactate dehydrogenase of cells and of the extra-cellular space revealed a similar extent of inhibition of both synthesis and leakage following exposure to D-galactosamine. Hepatocyte cultures that had been labeled before D-galactosamine treatment lost intracellular protein-associated radioactivity almost as rapidly as control cells up to the time of measurable enzyme leakage; thereafter, the rate of 3H-loss increased in the treated cells. Lactate dehydrogenase present in the medium is degraded less rapidly than the enzyme in the intracellular space. This explains the apparent increase of total lactate dehydrogenase activity in D-galactosamine-treated as compared to control cultures. Following [3H]leucine addition to D-galactosamine-treated cultures, the specific radioactivity of the leaked lactate dehydrogenase in the medium was never greater than that of the enzyme in the cytosolic compartment. The data rule out a direct excretion of newly synthesized enzyme as a result of D-galactosamine action.

The complete amino-acid sequence of alpha A- and beta-chains from the major hemoglobin component (HbA) of American Flamingo ( Phoenicopterus ruber ruber) is presented. The minor component (HbD) with alpha D-chains was detected in similar amounts (25%) as in chicken and pheasant hemoglobins. The comparison of American Flamingo and Greylag Goose (Anser anser) hemoglobins shows that alpha A-chains differ by 22 exchanges and beta-chains by only 4 exchanges. Two substitutions modify alpha 1 beta 1-contacts. Amino-acid replacements between American Flamingo and other bird hemoglobins are discussed.

Treatment with phenobarbital causes an increased urinary excretion of tetrahydroxylated bile acids in patients suffering from intrahepatic cholestasis. The main components were isolated from urine by means of column and thin-layer chromatography and were studied as methyl esters by nuclear magnetic resonance spectroscopy. The results obtained strongly support the contention that the main components are 1 beta-, 6 alpha- and 6 beta-hydroxylated derivatives of cholic acid.

The erythrocyte receptors for Vicia graminea (Vg) anti-N lectin have been investigated after 125I-labelling of the purified lectin and binding to membrane components separated by dodecyl sulphate polyacrylamide gel electrophoresis. GP alpha (synonym glycophorin A or MN glycoprotein) and GP delta (synonym glycophorin B or Ss glycoprotein) are the main Vg receptors of native human blood group NN and MN erythrocytes whereas Vg lectin only binds to GP delta from MM red cells. The glycoprotein of 28 kDa present in Mi III erythrocytes (a presumed variant of GP delta) carries Vg receptors. Both binding studies and agglutination experiments with this lectin suggest that the delta Mi III gene might produce more glycoprotein molecules than the normal delta gene. Binding of Vg lectin to hybrid glycoproteins [from Mi V, St(a+) and Dantu (+) donors] produced by unequal crossing-over between alpha and delta genes, may occur if the molecules exhibit N activity. The lectin does not bind to sialic acid- and galactose-deficient glycoproteins from Tn erythrocytes and no binding could be detected in the region of GP delta of erythrocytes from S-s-U-individuals. Addition of N-acetylgalactosamine residues to the alkali-labile oligosaccharides attached to GP alpha and GP delta, as found in Cad erythrocytes, decrease the binding capacity for Vg lectin. Finally the absence of Vg lectin binding sites on native GP alpha molecule from MgMg and McM erythrocytes, which carry well defined variants of this glycoprotein, supports the view that the binding site of the lectin on native glycoproteins is located at the N-terminal end of glycoprotein (GP alpha and GP delta) with N specificity (N-terminus = Leu).

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.