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Biomedical subjects

M Iwai

Publications and source records attributed to M Iwai.

At least 253 records · Page 14Linked to original sources
PubMed1982

Modification of carboxyl groups in Geotrichum candidum lipase.

Lipase from Geotrichum (Geo.) candidum was rapidly inactivated by incubation with water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC), or 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluenesulfonate (CMC), at pH 4.8. The pH dependence of the rate of inactivation was consistent with the modification of carboxyl groups in the lipase. Reaction of the lipase with EDC in the presence of the nucleophile taurine showed that about 9 carboxyl groups per molecule of enzyme were modified with concomitant total loss of activity. This number was reduced to 4 when CMC was used as a carbodiimide instead of EDC. The modification had no effect on the CD spectrum in the ultraviolet region. Kinetic analysis of the effect of CMC on the lipase indicated that at least 1 CMC molecule bound to the enzyme during inactivation.

CME-Carbodiimide↗
PubMed1981

Determination of serum unconjugated estrone, estradiol-17 beta and estriol during pregnancy by selected ion monitoring.

A simple, rapid and highly specific method by selected ion monitoring (SIM), using 9 alpha, 11 alpha-[2H2]estrone, [2,4-2H2]estradiol-17 beta and 2,4-[2H2]estriol as internal standards, was developed for the determination of serum estrogens during pregnancy. Serum samples were submitted to a simple extraction procedure and were analysed after formation of the trifluoroacetic anhydride derivative. The inter-assay coefficients of variation for estrone, estradiol-17 beta and estriol were 3.73%, 3.42% and 3.49%, respectively. The results obtained by SIM were compared with analysis performed using radioimmunoassay.

Estradiol↗
PubMed1981

Reversibility of the modification of Rhizopus delemar lipases by phosphatidylcholine.

Rhizopus (Rh.) delemar (ATCC 34612) lipase is modified by its binding with phosphatidylcholine (PC); such binding enhances the lipoprotein lipase (LPL) activity, shifts the isoelectric point (pI) to the acidic side and decreases its alpha-helical content ((1980) J. Biochem. 88, 533-538). The results of density gradient ultracentrifugation proved that PC binding to lipase molecule was depleted by the treatment of PC-bound lipase with 0.3% Triton X-100 and 0.1 M NaCl. By this treatment, LPL activity was decreased almost to the original activity. At the same time, alpha-helical content recovered to that of the original lipase and the isoelectric point recovered from pI 6.5 to nearly the pI of the original lipase. These data indicate that the modification of Rh. lipase by PC is reversible. Furthermore, the results of an experiment with 2-[1(-14) C]oleoyl PC showed that lipase having high LPL activity contained about 5 mol of PC per mol of lipase.

Isoelectric Point↗
PubMed1980

Purification and properties of partial glyceride hydrolase of Penicillium cyclopium M1.

A lipolytic enzyme which hydrolyzed monoacylglycerols more easily than triacylglycerols was found in the culture broth of Penicillium cyclopium M1. The enzyme was purified to homogeneity and its properties were investigated. Among various substrates used, monoacylglycerols, especially those of medium chain fatty acids, were hydrolyzed very rapidly. Although the rate was low, the enzyme hydrolyzed methyl esters of fatty acids, Span or triacylglycerols of medium chain fatty acids. Based on its substrate specificity, the enzyme was regarded as a partial glyceride hydrolase. When the partial glyceride hydrolase was used in conjunction with lipase on triacylglycerol, the degree of hydrolysis of triacylglycerol became extremely high.

Carboxylic Ester Hydrolases↗
PubMed1980

Modification of Rhizopus delemar lipase by its binding with phospholipids.

Rhizopus delemar (ATCC 34612) lipase was modified by phospholipid (PL)-treatment so as to enhance its activity on lipoprotein. In order to detect change in lipase conformation in the modified state, a preliminary experiment was performed to remove PL from the PL-treated lipase solution which included PL nonessential to enhancement of lipoprotein lipase (LPL) activity. It was found that treatment with a mixture of isopropyl ether: n-butanol (3 : 1) was suitable for this purpose because of the stability of the enzyme. Changes in isoelectric point and alpha-helical content of lipase caused by PL-treatment were studied by means of isoelectric focusing and circular dichroism spectrum. The isoelectric point of lipase was found to shift to the acidic side on its binding with phosphatidylcholine (PC) or cardiolipin (CL). The circular dichroism spectra of the original lipase and PL-treated lipases indicated that the alpha-helical content of lipase decreased on its binding with PL. In CL-bound lipase, which was more greatly enhanced as to LPL activity than was PC-bound lipase, alpha-helical content was decreased to a larger extent than that of PC-treated lipase.

Cardiolipins↗
PubMed1979

Synthesis of various kinds of esters by four microbial lipases.

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.

Aspergillus niger↗
PubMed1979

Low resolution crystal structure of lipase from Geotrichum candidum (ATCC34614).

Lipase from Geotrichum candidum (ATCC34614) is a glycerol ester hydrolase which has a molecular weight of 55,000 with about 7% carbohydrate, displaying a high affinity for triolein. The enzyme was crystallized from more than 2% protein solution without using any salt or organic solvent. The crystals were cross-linked by soaking in 0.37% glutaraldehyde solution (0.1 M acetate buffer solution, pH 5.6). The structure was determined by X-ray diffraction using the isomorphous replacement technique. Two heavy-atom derivatives [K2PtCl4 and UO2(CH3COO)2] were obtained by the soaking method. The electron density map calculated at 5 A resolution clearly showed the molecular boundary. A balsa wood model was made on the basis of the 6 A electron density map. The molecular has an ellipsoidal shape with dimensions of 70 A X 50 A X 50 A. Several columns of density corresponding to alpha-helix and a few clefts were found in the molecule. The active site is presumably located in the vicinity of one of the Pt sites in the Pt-derivative crystal, judging from the inactivation of the enzyme by K2PtCl4.

Cross-Linking Reagents↗
PubMed1977

Glyceride synthesis by four kinds of microbial lipase.

Apart from their usual mechanism of action, lipases from Aspergillus niger and Rhizopus delemar also catalyzed the synthesis of glycerides from oleic acid and glycerol. Lipases from Geotrichum candidum and Penicillium cyclopium were inactivated by oleic acid, but were stable in the presence of casein, albumin or buffer of appropriate pH. Lipases from Aspergillus niger and Rhizopus delemar synthesized glycerides from, not only fatty acid, but dibasic acids and aromatic acids, making ester bonds only at position 1 and 3 of glycerol. In contrast, lipases from Geotricum candidum and Penicillium cyclopium synthesized glycerides only from long chain fatty acids, and made ester bonds at all three available positions of the glycerol molecule.

Aspergillus niger↗