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1.
1. The hydrophobic N,N'-dicyclohexylcarbodiimide (DCCD) inhibits the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal fraction. 2. The inhibition is dependent on time and concentration, with half-maximal inhibition occurring at 0.4 mM concentration of carbodiimide after a 0.5 hr incubation at room temperature. 3. ATP does not protect against the inhibition. 4. Two water-soluble carbodiimides, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide (CMCD) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCD), are not inhibitory. 5. Inhibition of Mg(2+)-ATPase activity by DCCD is accompanied by covalent incorporation of the radioactive agent into the partially purified enzyme preparation.  相似文献   

2.
Summary Immunohistochemically, coagulation F-VIII-R:AG in vascular endothelial cells can be demonstrated with antihuman F-VIII-R:AG antisera after fixation with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, using both peroxidase and fluorescence techniques. The generally used aldehyde containing fixatives, result in a loss of immunoreactivity. Carbodiimide is preferable as a fixative for the cellular localization of F-VIII-R:AG.  相似文献   

3.
在连接试剂1-乙基-3(3-二甲基胺基丙基)碳二亚胺盐酸盐(1-ethyl-3(3-dimethylamino propyl)-carbodiimide,EDC)的作用下,将水相合成的量子点(Quantumdots,QDs)与人表皮生长因子(Epidermal growth factor,EGF)进行了连接,通过毛细管电泳、吸收光谱以及荧光光谱对连接前后的QDs进行了表征,获得的QDs—EGF可识别卵巢癌细胞SKOV-3。  相似文献   

4.
采用微波加热法制得15 nm的胶体金颗粒,并将小分子抗原氨苄青霉素分别以戊二醛和碳化二亚胺为偶联剂与牛血清蛋白偶联制成全抗原,再通过最佳标记量的确定分别将氨苄青霉素、戊二醛法全抗原、碳化二亚胺法全抗原同胶体金进行结合,红外检测和杯碟法的抑菌试验表明采用仅有戊二醛作为偶联剂的全抗原能够很好地与胶体金结合,并保持良好抗原活性。  相似文献   

5.
The affinity of human plasminogen for lysine-Sepharose is eliminated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, but presence of 6-aminohexanoic acid prevents affinity loss. The data indicate that abolition of affinity for lysine-Sepharose is due to reaction of carboxyl-group(s). 1,2-Cyclohexanedione modification of arginine residues of plasminogen also abolishes binding to lysine-Sepharose, and 6-aminohexanoic acid provides protection against the effect of the reagent. It is suggested that the essential carboxyl- and guanidino-groups bind the amino- and carboxyl function of ω-aminocarboxylic acids, respectively.  相似文献   

6.
A strategy suitable for the synthesis of larger peptides is proposed. It involves the following four considerations: (1) all of the side-chain functional groups are protected by benzyl-type protective groups; (2) a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, is used for the fragment-condensation reactions together with 1-hydroxybenzotriazole as the additive; (3) all the protective groups are cleaved simultaneously by the HF method in the final stage of the synthesis; and (4) side products formed are detected and removed by an efficient high-performance liquid chromatography procedure. The usefulness of these procedures is demonstrated taking the synthesis of human parathyroid hormone [hPTH(1–84)] as an example.  相似文献   

7.
Carbodiimide-dependent inactivation of dihydrofolate reductase   总被引:1,自引:0,他引:1  
Dihydrofolate reductase from amethopterin-resistant Lactobacilluscasei was inactivated by a water soluble carbodiimide, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HCl. The rapid inactivation observed at pH 5.0–6.0, coupled with lack of recovery of activity from inactivated samples incubated with NH2OH was consistent with modification of enzymic carboxyl groups. Significant protection against inactivation was provided by 7,8-dihydrofolate and NADPH. Analysis of the reaction order suggests that the carbodiimide-dependent inactivation may result from modification of a single essential carboxyl group.  相似文献   

8.
The aminoglycoside antibiotics such as neomycin, gentamicin, kanamycin and streptomycin stimulated the purified enzyme phosphatidylinositol-specific phospholipases C from Bacillus thuringiensis at pH 5.5. The involvement of net positive charge of aminoglycoside antibiotics (AA) on phosphatidylinositol-specific phospholipases C activation was probed by modifying the carboxyl group of Asp and Glu present in the enzyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC). Intrinsic Trp fluorescence of EDAC modified and unmodified PI-PLC in the presence of AA confirmed the interaction of AA with side chain carboxyl group of aspartic and glutamic acid of the enzyme. Thus, the possible interaction of aminoglycoside antibiotics with phosphatidylinositol-specific phospholipases C is predicted to be mediated through the aspartic and glutamic acid residue(s) of the protein.  相似文献   

9.
Polydiethylacrylamides (degree of polymerization, 13-470) containing a terminal carboxyl group were obtained by the method of radical polymerization of N,N-diethylacrylamide in the presence of mercaptoacetic acid. In the presence of 1-ethyl-(3,3-dimethylaminopropyl)-carbodiimide, these polymers reacted with ovomucoid to produce its polymeric derivatives. The values of the lower critical mixing temperature of these derivatives and the inhibitory activities of immobilized ovomucoid were determined by the length and amount of polydiethylacrylamide macromolecules bound to the molecule of ovomucoid.  相似文献   

10.
A new perfusion protocol was developed to detect histaminc-like immunoreactive neurons at the electron microscopic level. By stepwise perfusion of 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide and paraformaldchyde solutions, the brain block could be cut with a vibratome and the immunoreactivity could be detected using the avidin-biotin-peroxidase-complex method. We used this method to study the ultrastructure and synaptic relations of the histaminergic neurons in the postmanunillary caudal magnocellular nucleus of the rat hypothalamus. This method should also be useful for examination of histaminergic neurons in other tissues and the synaptic relations of histaminergic neurons with other neurotnunsmitter-containing neurons by double immunostaining.  相似文献   

11.
Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl3-phenol mixed solvent without danger of epimerization or of significant ester formationwith the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employedin the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine(HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl3-phenol were determined and successfully applied to the synthesis of amyloid -peptide (1-42), (1-43) and [Pyr3]-(3-42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra andfluorimetric assay.  相似文献   

12.
Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.  相似文献   

13.
Summary Segment condensation reaction of sparingly soluble protected peptides proceeded smoothly in CHCl3-phenol mixed solvent without danger of epimerization or of significant ester formation with the carboxyl component when 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was employed in the presence of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). The optimal conditions for enhancement of peptide coupling mediated by EDC/HOOBt in CHCl3-phenol were determined and successfully applied to the synthesis of amyloid β-peptide (1–42), (1–43) and [Pyr3]-(3–42). These peptides of high homogeneity were used to examine the relation between structure and amyloidogenesis by means of CD spectra and fluorimetric assay.  相似文献   

14.
Porcine pancreatic lipase was modified with Z-proline via the constitution of amide bonds between the free amino groups of lipase and the carboxyl groups of Z-proline, which were activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Different amounts of Z-proline were bound to lipase. Modification degree was determined by 2,4,6-trinitrobenzene sulphonic acid (TNBS), by means of the decrease in free amino groups on lipase. The reason for choosing Z-proline was its unique structural characteristics, protected amino groups, and its effect on protein conformation by reducing the flexibility of the lipase molecule, thus achieving stabilization against changes in pH and temperature.After the modification, porcine pancreatic lipase was found to have new physicochemical characteristics, such as optimum alkaline pH stability and thermal stability at elevated temperatures.  相似文献   

15.
Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.  相似文献   

16.
Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide. pKa of the modified carboxyl groups is approximately 6.5. An investigation of the initial steps of enzymatic catalysis monitored by a changes in the circular dichroism spectra and in an oxidation reaction with ferricyanide made it possible to conclude that the modification interferes with the donor substrate attachment to the enzyme. Evidence obtained was suggesting that the carboxyl group of the active center facilitates dissociation of a proton from the carbon atom in the second position of the thiamine pyrophosphate thiazolium ring.  相似文献   

17.
1. Of the 15 tyrosyl residues/subunit of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase) only one residue is specifically modified at pH 8.0 with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. 2. The acylation of this single tyrosyl residue leads to the loss of the enzyme activities (hexokinase and ATPase) by a first-order process, which can be fully reversed by treatment with hydroxylamine. 3. ATP does not protect the enzyme against chemical modification and inactivation; however, glucose exerts a noticeable though indirect protection effect against chemical modification and inactivation. 4. The chemically modified enzyme, purified by column chromatography, has 14% of the activity of the native enzyme, but the Km for ATP-Mg or glucose remains unchanged as does the pH optimum of activity. Results of conformational studies (ultracentrifugation, fluorescence, thermostability and chemical reactivity of the sulfhydryl groups) indicate that the decrease of enzyme activity due to the modification of the tyrosyl residue is related to a localized perturbation of the enzyme active-center region.  相似文献   

18.
Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. In both cases the kinetics of inactivation is biphasic, which agrees with the presence of two active centers in the enzyme molecule differing in their sensitivity to the inhibitors. There is some evidence that inactivation of transketolase is due to modification of carboxyl groups of enzyme. Complete inactivation is achieved by modification of one carboxyl per active site of the enzyme. The experimental results suggest that the carboxyl group is essential for the enzymatic activity of transketolase.  相似文献   

19.
In vitro "simultaneous processing" was investigated in which fibril formation of collagen and cross-linking occur simultaneously in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as a cross-linking reagent. Fibril formation in simultaneous processing was monitored using turbidity. The EDC in simultaneous processing increased T(1/2) (time required for half of the plateau value in turbidity) and decreased the degree of the fibril formation dose dependently. The reduced fibril formation rate (T(1/2) > 60 s) suggests the introduction of intrafibrillar cross-linking during fibril formation. The collagen gels prepared using simultaneous processing had a compressive modulus that was 6-fold higher than that using sequential processing, which is an advantage of simultaneous processing. Atomic force microscopy images acquired under water on the wet gels demonstrated that the simultaneous processing provided a unique double-network structure: intrafibrillarly cross-linked collagen fibrils among which nonfibrous collagens act as interfibrillar cross-linkages.  相似文献   

20.
Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.  相似文献   

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