Human inter-alpha-trypsin inhibitor has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G. The protein was cleaved into two major fragments without loss of activity by incubation with Serratia marcescens metalloproteinase, and these were separated by ion-exchange chromatography. Inhibitory activity was found in only one of the fragments, the amino-terminal sequence of which was found to be identical with that of the native protein, as well as with that reported earlier for the urinary trypsin inhibitor. It may thus be concluded that the reactive site of the inter-alpha-trypsin inhibitor is located in the amino-terminal region.
3-Hydroxyanthranilic acid was identified in lymphocyte cultures during allogeneic stimulation. The metabolite and the synthetic product have identical HPLC retention times, fluorescence spectra, fluorescence intensities as a function of pH value and electrochemical behaviour.
A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.
The hemocyanins of the horseshoe crab Limulus polyphemus (48-mer), the tarantula Eurypelma californicum (24-mer), and the lycosid spider Cupiennius salei (dodecamer, hexamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Among the subunits a to g of Eurypelma on the one side, and I to VI of Limulus on the other, a number of cross-reactions were obtained which agree with the topologic subunit positions in the published models of quaternary structure: a = II, b-c = V-VI, d = IV, e = I, f = IIIb, g = IIIa (IIa). However, cross-reactivity was only strong in the following combinations: a/II, d/IV, b-c/V-VI (the monomers of the two heterodimers could not be correlated individually). A rather weak cross-reaction was obtained in the case of e/I and g/IIIa (IIa); a cross-reaction between f and IIIb was almost undetectable. On the other hand, f/IV clearly cross-reacted, and so did e/IIIa (IIa), which apparently is not in agreement with the two models of quaternary structure. These unexpected relationships, however, indicate the possible phylogeny of the subunits. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunit IV of Limulus and, moreover, revealed common antigen determinants present on these subunits. Denaturation of hemocyanin subunits of the three species with 8M urea yielded a completely different immunological behavior in that in all intra- and interspecific combinations the reaction of immunological identity was obtained. The published models of quaternary structure and a possible subunit phylogeny of cheliceratan hemocyanins is discussed in view of the present results and the results of the preceding paper. [Markl, J. et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 619-631.]
In its natural environment the protist Dictyostelium discoideum grows on bacteria and queuosine-containing tRNAs of the bacteria serve as source of the nutrient factor queuine. This deazaguanine derivative is inserted into tRNAAsp, tRNAAsn, tRNAHis and tRNATyr of the amoebae. The axenic strain AX-2 of D. discoideum grows equally well in a defined medium with or without exogenous queuine. When queuine is omitted, changes occur in lactate levels, lactate dehydrogenase patterns and cytochromes and the amoebae cannot differentiate after a metabolic stress. In this report we show that growing cells contain two-fold higher levels of tRNAAsp and tRNATyr when sufficiently supplied with queuine, than those lacking queuine. In tRNAAsp a new, as yet unidentified, derivative of queuine has been discovered. When RNA synthesis is totally inhibited by actinomycin, tRNAAsp and tRNATyr remain stable in queuine-containing, but not in queuine-lacking cells. In contrast, tRNAAsn and tRNAHis become partially degraded in both conditions. We suggest that free queuine can be obtained from endogeneous tRNA and that differential salvage of queuine by tRNAs of the Q-family plays a role in the regulation of genes encoding components of redox chains.
The specificity of various allo- and autoantibodies, which agglutinate normal erythrocytes, but do not react with En(a-) red cells and normal erythrocytes, treated with trypsin (anti-EnaTS) or ficin (anti-EnaFS), was investigated. Various fragments and modification products of the major (MN) red cell membranes sialoglycoprotein were used in hemagglutination inhibition assays. Six anti-EnaFS sera were found to be directed against the residues approx. 46-56 of the molecule. Five of these require the carbohydrate unit, attached to Thr50, for binding. One anti-EnaTS serum was found to be directed against the residues approx. 36-42. Another antibody with anti-EnaTS specificity was shown to react with the residues 31-39 in some of the MN sialoglycoprotein molecules, namely those not glycosylated at a certain position (probably Thr33). A third anti-EnaTS serum, directed against the sequence domain around Lys30, was also found to react only with a fraction of the molecules, apparently due to the variable attachment of oligosaccharides in that region. The heterogeneity of glycosylation, detected by these two sera, appears to account for the partial tryptic and chymotryptic cleavage in this domain of the MN sialoglycoprotein, which has been described previously. Heterogeneity of the glycosylation at various positions of the molecule could be established by the isolation and analysis of peptides.
The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.
Two types of alpha- and two types of beta-chains are found in the hemoglobin of yak population. The complete amino-acid sequences of the four polypeptide chains were determined. The two alpha-chains differ by two and the two beta-chains by three amino-acid substitutions. The substitution of valine at position 135 in the beta II-chain may be responsible for the high intrinsic oxygen affinity of yak hemoglobin.
Three low molecular mass polypeptides have been isolated by using the technique of organic solvent extraction of thylakoid membranes or whole cells from Rhodopseudomonas viridis. Their primary structures were determined by long liquid phase sequencer runs, combined with the isolation and sequence analysis of the C-terminal o-iodosobenzoic acid fragment and carboxypeptidase degradation. The polypeptide which consists of 58 amino-acids and is 46% homologous to the antenna polypeptide B880-alpha from Rhodospirillum rubrum was designated as B1015-alpha (1 His residue). The sequence homology between the second polypeptide, named B1015-beta (55 amino acids, 2 His residues) and B880-beta from Rs. rubrum is 52%. For the third polypeptide consisting of 36 amino acids and exhibiting a high hydrophobicity, no equivalent polypeptide has so far been found in other purple bacteria. The molar ratio of these three organic solvent soluble polypeptides from Rp. viridis was estimated to be 1:1:1. Accordingly, the 36 amino-acid polypeptide is likely to be an additional constituent of the light-harvesting complex B1015, consequently termed as B1015-gamma. According to hydrophathy profiles, the transmembrane arrangement of B1015-alpha and B1015-beta within the thylakoid membrane is supposed to be similar. B1015-gamma, however, shows a somewhat different hydropathy profile. A particular feature of this polypeptide is its high amount of aromatic amino acids. It is postulated that B1015-gamma is involved in the formation of regular arrays of light-harvesting complexes.
The widely used fluorometric microsomal monooxygenase test for 7-ethoxycoumarin O-dealkylation was reinvestigated with regard to other possible hydroxylation products. By HPLC-analysis no beta-hydroxylation of the ethyl group and no 8-hydroxylation could be detected. Only a small percentage of 6-hydroxylation occurred, but as a new major metabolite 7-ethoxy-3-hydroxycoumarin was found in quantities depending on the microsomal preparation used. The ratio of O-dealkylation to 3-hydroxylation varied according to species, induction, buffer and pH, suggesting that different isozymes of cytochrome P450 were involved. The isozyme mainly responsible for 3-hydroxylation exhibited a great dependence on cytochrome b5 as the donor for the second electron. The fluorometric test does not include 3-hydroxylation due to the virtual absence of an emission spectrum above 450 nm.
Die Aktivität und zonale Verteilung von 4 Schlüssel-enzymen des Kohlenhydratstoffwechsels wurden in Lebern alloxan-diabetischer Ratten untersucht: Die Aktivität der glucogenen Phosphoenolpyruvat-Carboxykinase war auf 320
An estimation of 7,8-dihydro-6-hydroxylumazine in urinary samples of mice by high performance liquid chromatography on reversed phase material is presented. Urinary samples are diluted with water and oxidized by iodine. Chromatographic elution is done isocratically using aqueous buffer containing methanol. Effluent 6-hydroxylumazine is monitored by its fluorescence (382 nm excitation, 449 nm emission). The described method can be performed rapidly with satisfactory accuracy and precision. The electrochemical detection of 7,8-dihydro-6-hydroxylumazine was studied additionally. Total values of daily excreted 7,8-dihydro-6-hydroxylumazine of healthy and of Ehrlich ascites tumour-bearing mice were measured.
Hepatocellular injury was induced by exposure of primary cultures of rat hepatocytes to 4 mM D-galactosamine. The cell damage was very similar to that seen in vivo and in the isolated perfused rat liver, both in biochemical and in structural terms. The severity of the lesions caused by D-galactosamine was dependent on the age of the culture being treated. Less severe damage was found with older cultures. Since the primary metabolic effects of D-galactosamine were age-independent, the reduction in cell damage seems to be due to progressive cell dedifferentiation. Dexamethasone (1 microM) suppressed the full development of the injury, while 1 microM triiodo-L-thyronine enhanced it. A protection of hepatocytes by alpha 2-macroglobulin against the effects of D-galactosamine could be observed neither in vivo nor in vitro. Direct cytotoxic effects of endotoxin from Salmonella minnesota R 595 could be demonstrated only on hepatocytes in the early phases of primary culture using rather high doses of the purified lipopolysaccharide. It is unlikely that they play a major role in the hepatocellular injury seen following endotoxinemia in vivo. Lowering of extracellular Ca2+ concentration and additions of calcium/calmodulin inhibitors did not prevent cell injury after treatment with D-galactosamine. The results suggest that cell death is not due to an increased influx of Ca2+ into the cells.
Antibody 50S10.1 is a hybridoma-derived gamma 3 kappa antibody of BAB-14 mouse strain origin, with specificity for N-acetylglucosamine beta 1----3 linked to L-rhamnose, the immunodeterminant of the streptococcal Group A polysaccharide. The VL50S10.1 amino acid sequence is the fourth complete one reported with this specificity and the first fully determined V kappa 21A structure. Furthermore it is the first V kappa 21A isotype sequence derived from an antibody with known antigen specificity. The V kappa region of this and the previously described monoclonal anti-streptococcal Group A polysaccharide antibodies 7S34.1, 2S1.3 and 17S29.1 are compared, showing that in monoclonal antibody 50S10.1 a V kappa germline gene is expressed which is unrelated to those previously shown to be expressed in antibodies of this specificity. V kappa 50S10.1 increases the variability of known murine V kappa regions and confirms stretches of V kappa 21A sequences previously established.
The synthesis of gangliosides GM3, GM2, GM1, and GD1a solely lacking the fatty acid moiety, and thus called lysogangliosides in analogy to lysophospholipids, is described. Since a selective elimination of the fatty acid residue has not been achieved as yet, the gangliosides were first subjected to alkaline hydrolysis. By this procedure the fatty acyl as well as the acetyl groups of the sialic acid residue(s) were completely removed. The acetamido group of the N-acetylgalactosamine moiety of the gangliosides GM2, GM1, and GD1a was very little (congruent to 10%) hydrolyzed. In a two-phase system composed of water and ether, the selective protection of the sphingoid amino group was accomplished with a hydrophobic protective group (9-fluorenylmethoxycarbonyl). Lysogangliosides were obtained after re-N-acetylation of the sialooligosaccharide amino group(s) followed by removal of the protecting group. The overall yield was about 30%. The structures of the lysogangliosides were confirmed by chemical analysis as well as negative ion FAB mass spectrometry and 1H NMR spectroscopy. By simple re-N-acylation of lysogangliosides with any labeled fatty acid, labeled gangliosides are now obtainable that are identical with their parent gangliosides except for their labeled fatty acid residue. This has been demonstrated by the synthesis of GM1 with a [1-13C]palmitic acid moiety in its ceramide portion. If desired, double-labeled gangliosides may be obtained by use of labeled acetic anhydride in the synthesis of the lysogangliosides.
The plasma half-lives of glycosylated and unglycosylated alpha 1-proteinase inhibitor-radioactively labeled with [35S]methionine in rat hepatocyte primary cultures - were determined in the rat. Unglycosylated alpha 1-proteinase inhibitor was synthesized by hepatocytes in the presence of tunicamycin. Media from hepatocytes containing 35S-labeled glycosylated or unglycosylated alpha 1-proteinase inhibitor were injected into the tail veins of rats. At different times after injection alpha 1-proteinase inhibitor was isolated from plasma by affinity chromatography with anti-alpha 1-proteinase inhibitor Sepharose. Radioactivity measurements revealed a plasma half-life of 170 min for glycosylated alpha 1-proteinase inhibitor and of 30 min for the unglycosylated form of the inhibitor.
The concept of fractals as a dimensional measure to describe random irregularity and fragmentation, applied to the structure of gels enables the distribution of pore sizes to be explained as a generalized log-logistic function. This is achieved by introducing upper and lower limits to the power function of self-similar structured organization. In contrast to previous empirical relations for gel-permeation techniques, this approach provides a theoretically based relation between the measured distribution coefficients and the size of the eluting molecules as a measure without undue assumptions. Linearization by transformation of the cumulative log-logistic distribution function is proposed for regression of experimental data. The validity of the theory is demonstrated with size-exclusion data for different gel matrices. Other systems with properties based on irregularity and fragmentation of their heterogeneous structure could be described and analyzed by the same theory.
Protein–protein cross-linking has always been considered an important technique for elucidating the topography of ribosomal proteins within intact ribosomal particles. Most of the protein–protein cross-links described for the ribosomal subunits of Escherichia coli have been obtained using 2-iminothiolane as the cross-linking reagent. The powerful technique of protein–protein cross-linking for the analysis of the topography of ribosomal proteins within the ribosomal subunits has recently been criticized, since many of the results seemed incompatible with other topographical data. This chapter presents a new identification technique, using antisera against individual ribosomal proteins and immunoblotting for the unambiguous identification of the members of a cross-linked protein pair. This approach is especially valuable if non cleavable cross-linking reagents are being used. The results show that the migration behavior of some proteins—due to the cross-linking procedure—is altered during electrophoresis, whereas their antigenic properties remain unaffected. This alteration of the mobility of proteins would explain why the analysis of the members of a cross-linked protein pair solely on the basis of their electrophoretic mobility may easily lead to a misidentification of one or both of the components.
