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191.
猪精子凝集素的纯化,性质及其作用 总被引:3,自引:1,他引:2
用胎球蛋白-Sepharose亲和层析和凝胶过滤层析从精子和精浆中分离纯化了猪精子凝集素(简称BSL)。BSL的血凝活性只被若干糖蛋白和聚糖所抑制。BSL的分子量为56kd,由分子量分别为13.6kd(β)和16.0kd(α)的两个不同的亚基以α1β3所组成。BSL为糖蛋白,含中性糖3.2%,不含唾液酸。用ELISA法测定猪精子中BSL的含量及分布,表明70%嵌入在精子膜中,25%结合在精子表面, 相似文献
192.
The role of the primary amino groups of lysine sidechains in Ca2+ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca2+ and/or Mg2+ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca2+ and/or Mg2+. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca2+, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca2+ binding to the low affinity/high capacity Ca2+ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca2+ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca2+ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca2+ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca2+ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS
2,4,6-Trinitrobenzenesulfonic Acid
- GST
Glutathione S-Transferase
- SDS-PAGE
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
- EDTA
Ethylenediaminetetraacetic Acid
- EGTA
Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid
- MOPS
4-Morpholinepropanesulfonic Acid 相似文献
193.
Gudrun Borchert Sabine Bartel Inge Beyerdörfer Irmgard Küttner Laszlo Szekeres Ernst-Georg Krause 《Molecular and cellular biochemistry》1994,132(1):57-67
Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI2) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI2 (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical 1-adrenoceptor densities and affinities (as calculated from [3H]-CGP binding studies), Gi and Gs protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI2, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI2 is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection. 相似文献
194.
The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP
acyl-CoA binding protein
- BSA
bovine serum albumin
- CPT
carnitine palmitoyltransferase
- CPT0
malonyl-CoA sensitive CPT of the outer mitochondrial membrane
- CPT
malonyl-CoA insensitive CPT of the inner mitochondrial membrane
- OG
octylglucoside
- OMV
outer membrane vesicles
- IMV
inner membrane vesicles
Affiliated to the Department of Experimental Medicine, University of Montreal 相似文献
195.
Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH2 groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA
Bovine Serum Albumin
- CMC
Carboxy methyl Cellulose
- DTT
Dithiothreitol
- DMEM
Dulbeco's Modified Eagle's Medium
- DTNB
Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)]
- EDTA
Ethylenediaminetetraacetic acid
- FPLC
Fast Protein Liquid Chromatography
- FCA
Freund's Complete Adjuvant
- FCS
Fetal Calf Serum
- Gelonin-30
Gelonin modified by SPDP
- GnRH
Gonadotropin-Releasing Hormone
- Gelonin-SPDP
SPDP modified derivative of gelonin
- HEPES
(N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid])
- IFA
Incomplete Freund's Adjuvant
- 2IT
2-Iminothiolane
- IODOGEN
1,3,4,6-tetrachloro 3,6-diphenylglycouril
- oLH
Ovine Luteinizing Hormone
- oLH-SPDP
SPDP modified derivative of oLH
- oLH-10
oLH modified by 2IT
- oLH2IT
Molar ratio of oLH and 2IT
- PDP
2-Pyridyl-dithiopropionate
- PAP
Pokeweed Antiviral Protein
- RIP
Ribosome Inactivating Protein
- RP-HPLC
Reverse-Phase High Performance Liquid Chromatography
- RPMI
Roswell Park Memorial Institute
- RIA
Radioimmunoassay
- RRA
Radioreceptor Assay
- SPDP
N-Succinimidyl-3(2-pyridyldithio)propionate
- SDS-PAGE
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
- TCA
Trichloroacetic acid
- TFA
Trifluroacetic acid 相似文献
196.
197.
Hans-Jochen Schfer Gabriele Rathgeber Achim Schuhen Richard J. Berzborn 《FEBS letters》1994,340(3):265-268
UV irradiation of the ATPase (CF1) from spinach chloroplasts in the presence of 3'-arylazido-β-alanyl-8-azido ATP (8,3'-DiN3ATP) results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of -β cross-links. The results demonstrate an interfacial localization of the nucleotide binding sites on CF1. 相似文献
198.
Elementary Na+ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na+ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na+ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na+ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O1–O2 reaction (open(1) 1.4±0.3 msec; open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (open 3.0±0.5 msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP
o
significantly can interact with both types of noninactivating Na+ channels to reduce the dwell time in the conducting configuration. lodate-modified Na+ channels became drug sensitive during the O2 state. At –40 mV, for example, lidocaine reduced open(2) to 62±5% of the control without detectable changes in open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na+ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, alidocaine was 0.64×106 mol–1 sec–1 and adiprafenone 13.6×106 mol–1 sec–1. Trypsin-modified Na+ channels also appear capable of discriminating among these antiarrhythmics, the ratio adiprafenone/alidocaine even exceeded the value in iodate-modified Na+ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na+ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na+ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn. 相似文献
199.
The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca2+-transporting ATPases; cluster 2: Na+- and gastric H+-ATPases; cluster 3: plasma membrane H+-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K+, Cd2+, Cu2+ and an unknown cation). The one bacterial exception to this general pattern was the Mg2+-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques.
Correspondence to: M.H. Saier, Jr. 相似文献
200.
Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [3H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [3H]ifenprodil (40 nM) in a biphasic manner with IC50 values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC50 values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [3H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [3H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [3H]ifenprodil sites, defined either by ifenprodil displacement curves or by [3H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [3H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [3H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC50 of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [3H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [3H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [3H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist. 相似文献