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

An investigation of the mechanism in vitro of the non-oxidative segment of the pentose phosphate pathway using [5-14C]ribose 5-phosphate as a prediction labelling substrate with rat liver enzyme preparation is reported. Glucose 6-phosphate formed during the initial 0.5 h of reaction was heavily labelled in C-1 and thus is consistent with the prediction of the liver (L)-type pentose phosphate pathway (theoretically C-1/C-6 = 0.5). The reaction sequences of the fat (F-) type pentose phosphate pathway exclusively confine 14C to C-6 of glucose 6-phosphate. The presence of L-type reactions was further affirmed by the formation of D-arabinose 5-phosphate and D-glycero-D-ido-octulose phosphate which were identified and measured during the initial 2 h of incubation using gas liquid chromatography. After 3 h the concentration of 14C in C-1 of glucose 6-phosphate gradually declined and by 17 h of incubation the ratio of 14C in C-1/C-6 was 0.1. Possible reasons for the late changes in 14C-isotope distributions towards a pattern consistent with a contribution of F-type pentose phosphate pathway are given.

The self-association of glucose dehydrogenase (beta-D-glucose:NAD(P) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium was studied by analytical ultracentrifugation. The pH and composition of the buffer used were such that, owing to a reversible partial dissociation of the tetrameric enzyme, enzyme activity was reduced. It was found that under these conditions the protein exists in a monomer/dimer/tetramer association equilibrium.

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.

Antibodies 17S29.1 and 22S25.1 are monoclonal, hybridoma-derived gamma 3 kappa murine immunoglobulins with specificity for N-acetyl-glucosamine beta 1----3-linked to the L-rhamnose backbone structure, the immunodeterminant of the streptococcal Group A polysaccharide. The VL 17S29.1 amino-acid sequence is the third complete one reported from an antibody with this specificity, the second fully determined V kappa 25 structure and the first complete V kappa sequence of C57B1/6 origin derived from a carbohydrate-specific antibody. VL22S25.1 is a member of the V kappa 27 isotype of murine immunoglobulin VL regions. V kappa 17S29.1 and the determined part of the V kappa 22S25.1 sequence are compared to the previously described V kappa regions of streptococcal Group A polysaccharide-specific antibodies and to 12 selected partial and complete V kappa regions of antibodies with other specificities, predominantly to carbohydrate antigens. Both V kappa 17S29.1 and V kappa 22S25.1 increase the variability of known murine V kappa regions. They are the most homologous to the other V kappa regions derived from antibodies with streptococcal Group A polysaccharide specificity and share with them the amino-acid residue Arg74, so far characteristic for V kappa regions from antibodies with this specificity. The analysis of groups of independently expressed, highly homologous V kappa regions, namely V kappa 17S29.1 and V kappa 2S1.3 as one and V kappa 7S34.1 and V kappa 22S25.1 as a second group, offers the possibility of estimating the minimal number of V kappa germline genes involved in the immune response to the structurally defined streptococcal Group A polysaccharide antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

The elimination of aprotinin after intravenous infusion was exponential until 95% of the dose was cleared from the plasma after 1 h in dogs with bile-induced pancreatitis. The half-life of this part of the elimination was 10 min. The concentration of aprotinin in the peritoneal fluid reached a maximum plateau after 1 h. Direct intra-abdominal infusion of aprotinin was followed by a relatively slow elimination of the inhibitor from the cavity. One hour after the infusion the concentration of aprotinin in the peritoneal exudate was about 50% of the initial value. Four hours later the concentration of inhibitor with unchanged immunoreactivity and inhibiting capacity was still about 25% of the initial value. Based on the results of this experimental study an intraperitoneal dosage schedule for aprotinin was tested in three patients with haemorrhagic pancreatitis. A total amount of 14 X 10(6) KIU was given in repeated dosages during 18 h. This resulted in a minimum level of aprotinin in the peritoneal exudate of about 10 mumol/l. According to our earlier published data this level should largely block trypsin-induced effects relevant in pancreatitis. In conclusion; due to the rapid elimination of aprotinin from plasma, after i.v. application a therapeutically useful concentration is never reached in the peritoneum, while the elimination from the peritoneum is relatively slow, thus providing therapeutically useful concentrations which can be maintained for some time after i.p. application.

Very little is known of the metabolism of copper on a molecular level. For example, there is no evidence of an oxidative breakdown of Cu(I)-thionein leading to Cu(II). Thus it was of interest to use L- and D-amino-acid oxidases, amino oxidase and galactose oxidase to control the oxidation of Cu(I)-thionein by enzymically generated H2O2. In the presence of these enzymes Cu(II) was generated in each case. In a more detailed study the Cu(I)-thiolate chromophores of Cu-thionein were oxidized in the presence of xanthine oxidase as deduced from spectrometrical measurements using EPR and circular dichroism. Unlike Cu2Zn2-superoxide dismutase catalase inhibited the oxidative cleavage, suggesting peroxide as the actual oxidizing agent. Possibly there is an enzymic oxidative pathway for the generation of biologically important Cu(II).

The transient transcription of a rearranged mouse immunoglobulin kappa gene was studied in a monkey fibroblast cell line. The gene was inserted into an SV40 expression vector and the calcium phosphate coprecipitation method was used for transfection. The transcripts were correctly spliced; transcription, however, was initiated within the vector and not at the correct site 23-26 bp upstream of the gene, irrespective of the length of the upstream sequences (90, 160, 370, and 870 bp) in the plasmid constructs. In contrast, accurately initiated transcripts were observed when a plasmid containing the kappa gene with 870 bp of its upstream sequence was introduced into a lymphoid cell line; the plasmid was constructed from the pSV2-gpt vector and the electric impulse method was used for gene transfer in most experiments. Tissue-specific expression of kappa light chain genes in lymphoid cells is known to depend on the presence of an enhancer element in the J-C intron. The results reported in this paper suggest that the sequence elements pd and dc which are located upstream of the leader gene segment also act in a tissue-specific manner and that it is the initiation of transcription which is a tissue-specific event.

In a previous communication we described the preparation of the alpha/beta-chain complex of HLA-D membrane antigens from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1). In this paper we present the separation of the alpha- and beta-chains in quantitative scale using hydroxylapatite chromatography with a pH 7.15 phosphate buffer, containing sodium dodecyl sulfate. An alternative isolation procedure is electrophoresis, taking advantage of the different isoelectric points of the alpha- and beta-chains. The relative molecular masses, the isoelectric points and the N-terminal sequences are discussed and compared with the results of other investigators. Remarkable is the polymorphism of both the chains, especially the beta-chains. The importance of the previously described three step purification procedure for the preparation of these antigens for sequence studies has been pointed out.

The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.

The specificities of one viral and five bacterial sialidases were investigated by 1H-NMR-spectroscopy with substrates or substrate mixtures containing two sialic acid residues of different linkage types. This technique allows - in contrast to the methods used before - the simultaneous determination of the rates of hydrolysis of both NeuAc linkages in a single experiment. The substrate specificities of the enzymes are discussed on the basis of the relation of the rate constants k/k'. The data obtained are more exact and more informative than those of separate experiments as reported previously. Among the enzymes investigated, i.e. sialidases of fowl plague virus (FPV = VKH), Clostridium perfringens (CP), Vibrio cholerae (VC), Bifidobacterium bifidum var. pennsylvanicum (BBif), Bifidobacterium lactentis (BLac), and Arthrobacter ureafaciens (AU), the activity of the viral sialidase VKH shows the highest, the activities of the Bifidobacterium sialidases the lowest dependence on the nature and on the linkage type of the different substrates. All sialidases preferentially cleave the NeuAc alpha 2-3-Gal linkage with the exception of the enzyme of Arthrobacter ureafaciens (AU) which shows a higher affinity to alpha 2-6 linkages. However, this does not apply to the side-arm-linked NeuAc alpha 2-6 structure in NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6)-GlcNAc beta 1-3Gal beta 1-4Glc (Substrate B). This substrate in generally cleaved very slowly and is hardly affected by the viral enzyme. After the alpha 2-3 linkage, the alpha 2-8 bond in NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc(Substrate A) is most susceptible for the sialidases VKH, CP and VC. An elongation of the carbohydrate chain (Substrate D) is accompanied by a reduction of the rate of cleavage for all enzymes. The experiments with alpha 1-acid glycoprotein, fetuin, and with the glycopeptides obtained by proteolytic degradation of the latter, revealed the same specificity towards the alpha 2-3 and the alpha 2-6 linkages as the oligosaccharides. Influenced by the chemical nature and the size of the substrate, NeuAc is released from the native alpha 1-acid glycoprotein more quickly than from the corresponding glycopeptide. All sialidases investigated so far are strictly exo-enzymes as could be demonstrated by the cleavage of NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc (Substrate A).