In an attempt to provide immunological tools for subfractionation of high-density lipoproteins (HDL), monoclonal antibodies were raised against HDL complexes. Two clones identified a peptide, provisionally named K-45 (PI 4.5-4.9; molecular mass 45 kDa, range 42-48 kDa), whose plasma distribution and lipoprotein association were fully characterised. Gel filtration localised the peptide to the HDL region of human plasma where it co-eluted with apolipoprotein (apo) A-I, the structural protein of HDL. Complementary studies employing immunoabsorption with anti-(apo A-I) antibodies removed 90 % of K-45 from plasma: conversely, anti-(apo A-11) antibodies eliminated only 10 % of K-45. Immunoaffinity chromatography on an anti-(K-45) column revealed that the peptide was present in a distinct HDL subspecies containing three major proteins: K-45, apo A-I and clusterin or apo J. The lipoprotein nature of the bound fraction was indicated by electron microscopy (diameter 9.6 ? 3.3 nm) and quantification of lipids, the latter showing an unusually high triacyglycerol concentration. Plasma concentrations of K-45 were positively correlated with apo A-I and HDL-cholesterol and negatively correlated with apo B and total cholesterol. Thus, the peptide appears to be linked, directly or indirectly, to processes which give rise to an anti-atherogenic lipid profile. After completion of the present studies, an N-terminal sequence identical to that of K-45 was reported in recently isolated cDNA clones. These clones encode paraoxonase.Lipoprotein complexes are the principal transport vehicles for plasma lipids. As such, they are the focus of particular attention which derives from the designation of blood lipids as primary cardiovascular risk factors [l, 21. The rationale behind these studies is that a dysfunctional lipid transport system will be a major cause of the dyslipidaemias associated with premature cardiovascular disease [3]. In this context, an obvious pre-requisite is a comprehensive understanding of the normal functioning of the lipoprotein metabolic system ; unfortunately, this is not currently the case. It is due, in part, to the highly dynamic nature of this metabolic process, rendered even more intricate by extensive interactions between the major subclasses, very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoproteins [4]. The latter are of particular interest as they afford a measure of protection against cardiovascular disease [5, 61. Yet many aspects of HDL metabolism are poorly understood. Neither the origins nor the sites of catabolism of this lipoprotein species have been convincingly demonstrated. Additionally, the mechanisms by which HDL assure their postuCorrespondence to R.

Paraoxonase is a serum protein bound to high-density lipoproteins (HDLs). The physiological function of the enzyme is unknown, but a role in lipid metabolism has been postulated. To date, studies of the protein have had to rely on measurements of enzyme activity with various substrates. We have developed a highly specific, competitive e.l.i.s.a. using a previously characterized monoclonal antibody. The assay can detect 20 ng of paraoxonase with a working range of 75-600 ng. Intra- and interassay coefficients of variation were 6.5 and 7.9% respectively. Serum concentrations of paraoxonase in healthy subjects from Geneva and Manchester ranged from 25 to 118 micrograms/ml. There were significant differences in mean concentrations between the two groups (Geneva, 79.3 +/- 18.7 micrograms/ml; Manchester, 59.9 +/- 24.1 micrograms/ml: P < 0.001), differences also apparent when subjects were compared according to paraoxonase phenotype. These appeared to be largely a consequence of differences in apolipoprotein A-I concentrations between the two populations, suggesting that HDL particle number may be important in determining serum levels of paraoxonase. Paraoxonase specific activities were also significantly different between the two groups of subjects (Geneva, 2.08 +/- 0.96 units/mg; Manchester, 3.08 +/- 1.73 units/mg: P < 0.001), which may reflect differences in HDL particle composition. The e.l.i.s.a. should furnish the necessary complement to studies of paraoxonase enzymic activity and has already provided evidence for differences with respect to serum levels of the protein both between populations and between phenotypes within populations.

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (ginA, glnlI, and glnT

No abstract