Biological chemistry Hoppe-Seyler最新文献

An antibody that recognizes human nonpancreatic-type ribonuclease was obtained by immunizing a rabbit with a 14-residue synthetic peptide corresponding to the N-terminal sequence of eosinophil-derived neurotoxin which is identical to human liver ribonuclease. This amino acid sequence is unique to this protein. The anti N-peptide antibody was purified by protein A-Sepharose and by using ELISA and SDS-PAGE immunoblot techniques, the antibody reactivity against EDN and partially purified nonpancreatic-type ribonucleases from human plasma and urine was observed. Cross-reactivity with bovine pancreatic ribonuclease A and other proteins was not detected. In addition, the activity of the nonpancreatic-type ribonuclease was not affected by the antibody. The immune response was elicited without the need for a carrier protein showing that the N-terminal sequence of nonpancreatic ribonuclease contains a specific epitope. This antibody can be used for the immunological identification of both the native and denatured forms of this type of enzyme.

Clostridium perfringens produces two sialidases, one of which has a molecular mass of 71 kDa and is secreted, while the 'small', 43 kDa isoenzyme remains in the cells. The secreted, higher molecular mass sialidases of two different clostridial strains, DSM756T and A99, exhibit maximum activity at pH 5.5 and at 51 or 55 degrees C, respectively. The molecular mass of both enzymes is 71 kDa in SDS-PAGE and 63 kDa as determined by gel-filtration, which indicates the absence of subunits. Natural sialidase substrates are hydrolyzed at comparably high rates, e.g. the glycoproteins fetuin and bovine submandibular gland mucin, the homopolymer colominic acid, and the ganglioside mixture from bovine brain. The partially purified 'small' isoenzyme from C. perfringens A99 cells had similar properties to the corresponding recombinant sialidase isolated from the Escherichia coli host. It is located inside the clostridial and E. coli cells and exhibits maximum activity at pH 6.1 and 37 degrees C. A relative molecular mass of 32,000 was found with FPLC gel-filtration chromatography, while primary structure analysis yielded a value of 43,000. It differs a significantly from the 'large' isoenzyme by substrate specificity. Preferred substrates are oligosaccharides, while other, more complex sialoglycoconjugates are hydrolyzed only at very low rates. alpha 2,3-linkages are hydrolyzed much faster than alpha 2,6-bonds.

The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and N-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzymatic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH2, the subfractions were characterized by 1H NMR spectroscopy. A total of six oligosaccharides, ranging from Man4GlcNAc2 to Man9GlcNAc2 is present, Man6GlcNAc2 representing the most abundant one with 57% of all oligosaccharides.

A kinetic analysis of the mechanism of autocatalytic activation in the presence of a reversible inhibitor is presented. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. We have extended the kinetic equations derived to a particular case in rapid equilibrium conditions. This analysis is illustrated by the experimental study of the inhibition by p-aminobenzamidine of trypsin activity in its action on trypsinogen. In such system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The results obtained show that the apparent activation rate constant decreases non-linearly with the initial concentration of inhibitor, according to the equations obtained in the kinetic analysis.

ANP-receptors affinities (KD) and capacities (Bmax) were assayed in cryosections of glomeruli from 'malignant' hypertensive rats (2K-1C) and spontaneously hypertensive rats (PHR). Plasma ANP concentration was twofold higher in 2K-1C (P < 0.05) and PHR (P < 0.02) than in the respective controls, KD and Bmax for rANP99-126 and ANP103-123 did not differ. ANP mediated cGAMP release in 2K-1C rats was also unaffected. ANP-C glomerular receptors (i.e. displacement of tracer binding with ANP103-123) were not down-regulated and had unchanged peptide binding affinity in either kidney of rats with 'malignant' hypertension and in PHR. The difference between Bmax for rANP99-126 and Bmax for rANP103-123 (ANP-A receptor binding) indicates moderate up-regulation of ANP-A receptors in the clipped, and down-regulation in the contralateral kidney of 2K-1C (2K-1C, right vs. left, P < 0.05). Since [ANP]pl, and also Bmax and KD for ANP were similar in both hypertension models investigated, changes of the [ANP]pl/ANP-receptor system can not completely explain the marked natriuresis of rats with 'malignant' hypertension.

A full-length cDNA of the serum albumin (CSA) of the cobra (Naja naja kaouthia) was cloned from a lambda gt 11 library. It encodes a mature protein of 614 amino-acid residues homologous to the precursor of mammalian serum albumins. The 1 degree and 2 degrees structures of the CSA resemble those of the human variety. The putative toxin binding sites are mainly located in the subdomains IIA and IIIA. The relation between structural homology and function of the serum albumins (SA) is discussed. An analysis of their evolutionary tree revealed that anti-toxicity arose by < 90 amino-acid exchanges. The rate of substitution is much higher in the SA than in cytochrome C, which probably reflects the difference in evolutionary driving forces. The evolutionary period of the SA (6.7 +/- 0.1 M.Y.) significantly exceeds that of hemoglobin (5.8 M.Y.). Eight tripeptides in the nicotinic acetylcholine receptor (ACR), all flanking the putative toxin binding site, are also found in the CSA where they join to form 1 octa-, 1 penta- and 4 tripeptides, thus indicating the concerted evolution of two functionally linked proteins: toxin and antitoxin (CSA).

Incubation of progelatinase B, isolated from human polymorphonuclear leukocytes, with TIMP-1 leads to the formation of the progelatinase B/TIMP-1 complex. This complex behaves like a Janus in a similar manner as we previously described for the progelatinase A/TIMP-2 complex. It shows the properties of TIMP-1 and is a better inhibitor for gelatinase A than for gelatinase B. Treatment with trypsin leads to activation of the binary complex. The activity, however, amounts only to slightly more than 10% of the activity of free gelatinase B, not complexed with TIMP-1. When the progelatinase B/TIMP-1 complex inhibits an active matrix metalloproteinase, a ternary complex is generated that after activation displays a distinct higher proteolytic activity than the active binary complex. The active binary complex cannot be transformed into the active ternary complex.

The aim of the study was to investigate the impact of operant conditioning on the receptor pattern in the visual cortex of calves. A reward paradigm was used to induce conditioned preference for colours. Binding sites in visual cortex specimens from conditioned and naive animals were assayed in vitro on cellular basis after dissociation of the tissue by collagenase, incubation with fluorescent ligands and flow-cytometry for fluorescence analysis. The cellular counts were subdivided according to sedimentation at 200 g as well as via the flow-cytometrical histogram by size and granularity. Binding sites of dopamine D1 and D2 receptor subtypes, glucocorticoids, opioids, casein as well as glycine and N-methyl-D-aspartic acid (NMDA) were detected by fluorescent molecular probes. In displacing naloxone fluorescein from NMDA- and mu-opioid receptors NMDA and meth-enkephalin were used. Comparisons between portions of fluorescent cellular counts from visual cortex tissue of conditioned and naive animals revealed a small increase (1.2-fold, P < 0.05) in opioid receptors of large and high granulated counts, bearing > 80% D1 receptors, and a decrease (0.70-fold, P < 0.05) in less granulated counts with variable portion of D1 receptors. Conditioning resulted in higher and lower displacing rates by meth-enkephalin and NMDA, resp., and in a reduce in counts with dopamine D1 (0.8-fold, P < 0.05), glycine and glucocorticoid binding sites (0.6-fold in both cases, P < 0.01). A tendency of elevated phagocyte marker expression occurred in high granulated counts. The data suggest that conditioning is accompanied with significant and in part marked change in binding sites studied. Induction of scavenger activity may parallel this process. As cellular portion with glycine and glucocorticoid receptors were most markedly altered by conditioning, the neurochemical needs of the used paradigm seem to focus to motility-related functions.

Posttranslational import of preproteins into mitochondria has been reported to be inefficient in a homologous yeast in vitro system, suggesting a requirement for coupling of protein synthesis and import. We have characterized a homologous yeast in vitro system which allows posttranslational mitochondrial import of preproteins. The efficiency is comparable to that of the heterologous system with rabbit reticulocyte lysate and isolated yeast mitochondria. Import in the homologous system depends on mitochondrial surface receptors, a membrane potential and the matrix heat shock protein Hsp70. Import is not blocked by the protein synthesis inhibitor cycloheximide, but is impaired by induction of stable folding in preproteins. Our studies demonstrate a posttranslational translocation mechanism in the homologous system, strongly supporting the validity of conclusions drawn from the widely used heterologous import system.