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

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.

From the prokaryotic microorganism Acholeplasma laidlawii the major manganese-containing superoxide dismutase has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular mass of the enzyme was found to be 41 500 Da. It consists of two subunits of identical size and has an isoelectric point of 6.4. The enzyme contains 0.51 +/- 0.05 atoms of manganese per subunit. Its amino-acid composition and light absorption spectra are presented and compared with Mn- and Fe- containing superoxide dismutases from other prokaryotic organisms.

Employing a sensitive semi-quantitative electrophoretic-technique on acid-deproteinized serum, we found two previously unrecognized trypsin inhibitors migrating as beta 2- and gamma-globulins, respectively. The two inhibitory bands were also detected in native serum. They were not seen in 3 healthy persons, but were found in patients with uremia, cancer, inflammatory diseases and collagenosis. Immunological investigations showed no cross-reaction with antibodies against seven well-known proteinase inhibitors. The two trypsin inhibitors also inhibited pancreatic elastase.

The influence of the flavonolignane Silibinin on the rate of RNA synthesis in rat livers was studied in detail and the time course of the stimulatory effect was determined: 8 h after i.p. application a maximal increase of about 60% in nuclear RNA synthesis can be observed. The analysis of the RNA by electrophoresis on agarose and by sucrose gradient centrifugation demonstrated that in particular the ribosomal RNA (28S, 18S, 5.8S) synthesis is accelerated followed by enhanced incorporation of rRNA into mature ribosomes. During stimulation also changes in the pattern of 45S RNA can be observed. The synthesis of mRNAs, 5S RNA and tRNAs is not influenced by Silibinin, which was shown after separation of these moieties on oligo(dT)-cellulose, and by polyacrylamid electrophoresis, respectively. The clinically observed enhancement of liver cell regeneration during Silibinin treatment thus can be explained by an increase of the protein synthetic apparatus.

The concentration of a low molecular mass bronchial mucus inhibitor of proteolytic enzymes (BMPI) was measured in lung secretions from patients with chronic bronchitis and compared to the concentrations of albumin and alpha 1-proteinase inhibitor (alpha 1-PI). The concentration of all three proteins was lower in bronchoalveolar lavage samples than in sputum obtained from the same patient. However the relationship between the proteins (concentrations relative to each other) was similar in both secretions suggesting the differences in concentrations were only dilutional. The molar concentration of BMPI in both secretions was generally greater than that of alpha 1-PI suggesting that most of the anti-elastase screen (congruent to 75%) was due to the former protein. Furthermore inhibitory studies using both purified leukocyte elastase and porcine pancreatic elastase show that the inhibitory capacity of lung alpha 1-PI varied and accounted for about 16% of the total inhibition of leukocyte elastase in sputum. However, the inhibitory function of alpha 1-PI was less in bronchoalveolar lavage fluids compared to sputum (p less than 0.05) and accounted for about 5% of the total inhibitory capacity for leukocyte elastase. It is concluded that alpha 1-PI contributes only a part of the anti-elastase screen of secretions from patients with chronic bronchitis. Furthermore it is inactivated to a varying degree in most secretions and its remaining inhibitory capacity represents only a minor proportion of the total inhibitory capacity.

In situ reaction of erythrocyte membranes with dicarboxylic anhydrides leads to solubilization of hydrophobic integral proteins. Removal of peripheral proteins and bulk lipid by appropriate sedimentation and dialysis steps yields hydrophilic band 3 protein derivatives. These acyl compounds display size heterogeneity upon gel filtration. A chromatographically homogeneous acyl band 3 protein is obtained if the acylation is conducted in the presence of detergent and the detergent subsequently removed. Hydrophilic acyl derivatives of band 3 protein can be subjected to conventional analytical techniques without the use of detergents.

Various proteolytic fragments from the central region of the fibronectin subunit chains containing the main cell-affinity site were applied in cell binding studies using peritoneal macrophages of guinea pigs. A 125I-labelled 23-kDa peptide was relatively well bound by the cells. Attachment to cells was partially inhibited by wheat germ lectin, suggesting a lectin-like site in the cell-binding domain which recognizes oligosaccharide groups with terminal N-acetylglucosamine or N-acetylneuraminic acid. Binding was inhibited by N-acetylneuraminic acid with half-maximal effect at 2 X 10(-3) M. Other inhibitors were a sialic acid rich ganglioside preparation and fetuin, a sialic acid-containing glycoprotein. In contrast to the 23-kDa peptide a 125I-labelled 125-kDa fragment was only weakly bound, although it included the sequence of the 23-kDa peptide on its C-terminus. The residual binding was weakly inhibited by low concentrations of wheat germ lectin and was remarkably improved by higher concentrations. The behavior of the peptide was explained by the presence of a sialic acid-containing oligosaccharide side chain localized outside of the 23-kDa region and interacting with the lectin-like site in the cell-binding sequence. In accord with this suggestion a 95-kDa fragment representing the oligosaccharide-containing part of the 125-kDa peptide was capable of inhibiting at least partially the cell attachment of the 23-kDa piece. The results indicate a lectin-like affinity site in the cell-binding region of fibronectin which is accessible in the 23-kDa peptide, but is masked in the 125-kDa fragment and in fibronectin by a sialic acid-containing oligosaccharide moiety.

A radioactive photosensitive insulin analogue, 125I-N epsilon B29-(4-azido-2-nitrophenyl-acetyl)insulin, was covalently bound to the receptors of isolated rat adipocytes by irradiation with UV light. This caused a stimulation of lipogenesis. The relative potency of the covalent complexes to that of normal reversible complexes was calculated by comparing the amounts of radioactivity required to be covalently or reversibly bound by adipocytes to cause the same levels of stimulation. For several different occupancies , this relative potency was constant at 50 +/- 3%. Previous studies had shown that the relative potency of covalently bound 125I-N alpha B2-(4-azido-2- nitrophenylacetyl )des- PheB1 -insulin was only 25 +/- 4% under identical conditions. This demonstrates that the sites of crosslinking have a marked effect on the potency of the covalent hormone-receptor complex. It appears that attachment through the C-terminus of the B-chain leads to a better stabilization of the biologically active form than linking through the more flexible N-terminus.

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.

Arterial smooth muscle cells cultured from rat aorta were labelled with sodium [35S]-sulfate in combination with either [3H]glucosamine or [3H]mannose. The newly synthesized hyaluronate and sulfated proteoglycans obtained from the growth medium (M-PG) and extracted from the cell layer (C-PG) with 4M guanidinium chloride in the presence of proteinase inhibitors were purified by sequential fractionation on Sepharose 4B CL, equilibrium density gradient centrifugation and ion exchange chromatography under dissociative conditions. Gel filtration of M-PG resulted in the separation of free hyaluronate and two size classes of [35S]-proteoglycan populations eluted at Kav 0.15 (fraction M-A) and 0.48 (fraction M-B). On further fractionation M-A dissociated into hyaluronate (Mr 1.6 X 10(6)) and a proteoglycan monomer (M-PG A, Mr 180 000), which contained chondroitin 4-sulfate (Mr 21 000) as the main glycosaminoglycan moiety. The proteoglycan isolated from M-B (M-PG B) was identified as a proteoglycan monomer (Mr 200 000) containing mainly chondroitin sulfate/dermatan sulfate hybrid side chains (Mr 34 000). [3H]Mannose labelling and binding to ConA Sepharose of both M-PG A and B indicated the presence of oligosaccharides of the glycoprotein type. An analogous fractionation of proteoglycans associated with the cell layer yielded two hyaluronate-proteoglycan complexes (C- PreA and C-A). The proteoglycan monomers of these complexes (C-PG PreA and C-PG A) had Mr values of 420 000 and 130 000. A non-complexed proteoglycan monomer C-PG B (Mr 90 000) was also found. All cell layer bound proteoglycans had glycosaminoglycan side chains with Mr approximately 36 000 but the predominant glycosaminoglycan component was either heparan sulfate, chondroitin sulfate or dermatan sulfate. All cell layer bound proteoglycans contained [3H]mannose radioactivity, about 15% of which was bound to ConA Sepharose.