A low molecular weight compound, which inhibits the G1-S transition in rat hepatocytes, was obtained by tryptic hydrolysis of human alpha 2-macroglobulin followed by ultrafiltration at pH 10. It was purified by high-performance liquid chromatography on mu Bondapak C18 and mu Bondapak NH2 with a practically quantitative yield; from 5.1 g of alpha 2-macroglobulin, 2.8 micrograms of purified compound were recovered. Inactivation by specific enzymes and chemical analyses showed that the inhibitor is a sialylated glycopeptide whose peptide moiety contains a pyroglutamyl residue. Its molecular mass, estimated by gel permeation chromatography, would be in the interval 3,500-4,600. However, amino acid analyses indicated that it is not yet pure. All these data suggest that alpha 2-macroglobulin could be the carrier of the precursor form of the glycopeptide. 相似文献
Dopamine (DA) and cholecystokinin octapeptide carboxy-terminal (CCK-8) have been found to coexist in some mesolimbic neurons. The present investigation was undertaken in order to study the biochemical and behavioral interactions between CCK-8 and some central monoaminergic pathways. The action of the sulfated form of CCK-8 (10 micrograms/10 microliter intracerebroventricularly) on DA turnover in nucleus accumbens, olfactory tubercles and corpus striatum of the rat was determined after DA synthesis inhibition with alpha-methyl-p-tyrosine (250 mg/kg i.p.). Also, CCK-8 action (1-30 micrograms intracisternally) on DA synthesis was assessed by measuring accumulation of dihydroxyphenylalanine (DOPA) after DOPA-decarboxylase inhibition with NSD-1015 (m-hydroxybenzylhydrazine, 100 mg/kg i.p.). The contents of DA and its main metabolites, dihydroxyphenylacetic acid and homovanillic acid, together with serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in different brain areas after direct injection of CCK-8 into the ventral tegmental area (A10) or nucleus accumbens. Further, the effect of CCK-8 on amphetamine-induced locomotion and apomorphine-induced stereotypies was studied along with changes in spontaneous locomotion and rearing after CCK-8 injection into the ventral tegmental area and nucleus accumbens. No consistent statistically significant effects of CCK-8 on biochemical or behavioral assessments on measures of DA function were observed. However, injection of high doses of CCK-8 into the ventral tegmental area significantly decreased levels of 5-HIAA in the nucleus accumbens, olfactory tubercles and striatum. 相似文献
The linkage pattern of the K6-antigen was investigated using material from the urinary pathogen, LP 1092. The polysaccharide consists of ribose and 3-deoxy--2-octulosonate (KDO) in a ratio of 2:1. Colorimetric procedures, Smith degradation, methylation analysis, and nuclear magnetic resonance spectroscopy were applied to the whole polysaccharide and to a trisaccharide “repeating unit” obtained by mild-acid catalyzed hydrolysis. Together, the data are compatible only with a branched chain structure …… 相似文献
Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M4 and ≈2700 for the H4 inhibitor were calculated. An apparent Ki of ≈3 × 10?5 mM for the H4 and ≈7 × 10?5 mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k1 for the dimerisation step of M4 = 2.0 × 105 M?1 · s?1 and of H4 = 8.2 × 104 M?1 · s?1; k2 for the tetramerisation step of M4 = 2.8 × 105 M?1 · s?1 and of H4 = 1.2 × 105 · M?1 · s?1, were calculated, the second step still being the faster one. 相似文献
Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma. 相似文献
A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells. 相似文献
The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases. 相似文献
The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b6, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions. 相似文献
The binding of radiolabeled epidermal growth factor (EGF) to immobilized A-431 target cell membranes coupled to polyvinyl chloride microtiter wells is described. Saturation curves and Scatchard analysis of the data indicate that the observed binding parameters are consistent with those previously reported. Binding capacity of the membranes are approx. 6.6 pmol EGF per mg membrane protein. Kinetics of 125I-EGF binding were slower, however, than reported for binding to membranes in suspension, although binding constants were not greatly different. The high- and low-affinity binding constants for 125I-EGF were calculated to be approximately 1 · 1012 M?1 and 2.5 · 109 M?1, respectively. Application of this technique in a competitive binding assay requires no more than 2.5 μg of membrane protein per assay, is essentially complete after 60 min, and facilitates screening of a large number of samples in a short time. Therefore, this will assist in the evaluation and quantitation of EGF and EGF-related transforming growth factor activity in physiological fluids. This technique may also be applied to analyses of other hormone-receptor systems. 相似文献