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排序方式: 共有157条查询结果,搜索用时 15 毫秒
1.
2.
Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions. 相似文献
3.
Irving W. Wainer John C. Chen Heli Parenteau Sami Abdullah Julie Ducharme Hiltrude Fieger Joanna Iredale 《Chirality》1994,6(4):347-354
The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc. 相似文献
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Juan Sanchez-ramos Ihsan M. Diab Bruce Wainer Robert J. Dinerstein Lloyd J. Roth 《Biotechnic & histochemistry》1978,53(3):163-167
The localization of 3H-opiatcs in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ilial sections to dried photographic emulsion avoids this source of error. 相似文献
6.
Enantioselective HPLC methods have been developed for the resolution of (RS)-2-phenylcyclohexanone (compound 1) and (RS)-2-phenyltetrahydropyran-4-one (compound 4) and the diastereoselective and enantioselective separations of their respective cis- and trans-alcohols; reduction of compound 1 yields trans- and cis-2-phenyl-1-cyclohexanol (compounds 2 and 3, respectively) and reduction of compound 4 yields trans- and cis-2-phenyl-tetrahydropyran-4-ol (compounds 5 and 6, respectively). Compounds 1, 2, and 3 were stereochemically resolved using a chiral stationary phase (CSP) based upon amylose tris(3,5-dimethylphenyl carbamate) coated on 10 μm silica-gel (Chiralpak AD-CSP). Compounds 4, 5, and 6 were stereochemically resolved on a coupled column system where a column containing a CSP based upon cellulose tris(3,5-dimethylphenyl carbamate) coated on 5 μm silica (Chiralcel OD-H-CSP) was coupled in series to the AD-CSP. The strategy employed in the identification of the peaks in the respective chromatograms is also discussed in this presentation. Chirality 8:551–555, 1996. © 1997 Wiley-Liss, Inc. 相似文献
7.
Norepinephrine is N-methylated to epinephrine by the catalytic effect of the terminal enzyme in catecholamine biosynthesis, phenylethanolamine N-methyltransferase (PNMT). PNMT has been covalently immobilized onto a silica-based liquid chromatographic support, glutaraldehyde-P (Glut-P). The resulting PNMT-Glut-P stationary phase (PNMT-SP) was enzymatically active, stable, and reusable. Standard Michaelis-Menten kinetic studies were performed with both free and immobilized PNMT and known substrates and inhibitors were examined. The results demonstrate that the PNMT-SP can be utilized for the rapid screening of potential PNMT substrates as well as the screening of compounds for PNMT inhibitory activity. 相似文献
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9.
Markoglou N Hsuesh R Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,804(2):295-302
Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties. 相似文献
10.
Shibukawa A Ishizawa N Kimura T Sakamoto Y Ogita K Matsuo Y Kuroda Y Matayatsu C Nakagawa T Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,768(1):177-188
Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective. 相似文献