Selective 31P(1H) nuclear Overhauser effect study on the polar headgroup conformation of phospholipids in micelles in organic solvents

The polar headgroup structure of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in inverted micelles in chloroform or benzene was investigated by the selective 31P(H) nuclear Overhauser effect (NOE). In the frequency dependence of the 31P(1H) NOE, PC micelles in CDCl3 showed two maxima. The larger maximum was located at the resonance of the glycerol-CH2OP protons and the smaller at the resonance of the N-methyl protons. In PC/PE mixed micelles in C6D6, both PC and PE showed three maxima which were located at the resonance of the CH2OP protons, the N-methyl protons and the amino protons in the frequency dependence of the 31P-NOE. The N-methyl protons of PC and the amino protons of PE were closely spaced to the phosphate groups of neighboring lipid molecules. The polar headgroups of PC and PE in the mixed micelles were concluded to lie in the plane perpendicular to the molecular axes. The frequency dependence of the 31P(H) NOE for PS micelles in C6D6 showed the maxima at the resonances of the amino protons and the CH2OP protons. The polar headgroups of PS molecules were not extended parallel to the molecular axes in the inverted micelles.     

400 MHz proton nuclear magnetic resonance studies of the polar headgroup conformation of inositolphospholipids

400 MHz ¹H-resonances of 1-phosphatidyl-myoinositol (PI), PI 4-phosphate (DPI) and PI 4,5-bisphosphate (TPI) in CD₃OD were assigned. Proton resonances in the inositol moiety shift downfield with the increase in the number of the phosphate from PI to TPI. From the ¹H-¹H and ¹H-³¹P vicinal coupling constants, rotamer populations around bonds in the polar headgroups were calculated. The H-C(5)O-P bond at position 5 of the inositol moiety tends to assume a gauche form. The H-C(1)O-P and the H-C(4)O-P bonds are not so strongly restricted to the gauche form as the H-C(5)O-P bond. The conformation of the glycerol moiety in PI, DPI and TPI is similar to that in phosphotidylcholine (PC) and phosphatidylethanolamine (PE). The CH-CH₂O-P bond in the glycerol moiety assumes a trans form. The acyl chains prefer a gauche arrangement to each other around the CHCH₂OCOR bond.     

Hydrolysis of succinyl-trialanine p-nitroanilide by two enzymes associated with human high-density lipoproteins

Succinyl-trialanine p-nitroanilide (Suc-Ala3-pNA), a synthetic substrate for the determination of elastase activity, was hydrolyzed in sequence by two enzymes that were found to be associated with human high-density lipoproteins. The enzymes involved in the sequence of reaction were separated by ion-exchange chromatography from apo-lipoprotein A-I and A-II, major apoproteins of high-density lipoproteins. One, designated as fraction MK, cleaves Suc-Ala3-pNA to succinyl-dialanine and alanine p-nitroanilide (Ala-pNA), and the other, designated as fraction U, cleaves Ala-pNA to alanine and p-nitroaniline. Fraction MK was inhibited by dithiothreitol, EDTA, and 1,10-phenanthroline, whereas fraction U was inhibited by 1,10-phenanthroline and bestatin. In addition to these findings, fraction MK also hydrolyzed 2,4-dinitrophenyl-prolyl-glutaminyl-glycyl-isoleucyl-alanyl-glycyl-glutaminyl- arginine (DNP-octapeptide), a specific substrate for the determination of vertebrate collagenase. Neither native elastin nor native collagen was hydrolyzed by a mixture of the two enzymes. Fraction U was very similar to aminopeptidase M with respect to its enzyme characteristics studied.     

Polymorphisms of apolipoprotein C-III in two cases with sialidase deficiency

Since sialidase is thought to be one of the enzymes which are concerned with the polymorphic forms of apolipoprotein C-III depending on the contents of terminal sialic acid, the polymorphic forms of apolipoprotein C-III of very low density lipoprotein and apolipoprotein C-III levels were studied in two cases of sialidosis due to sialidase deficiency with or without β-galactosidase deficiency. The diagnosis was established by the defect of those enzymes in the leucocytes and cultured fibroblasts. But, no significant differences in polymorphic form of apolipoprotein C-III were observed between these two patients and controls.