Studies on the polymorphism of human apolipoprotein A-I.

Upon preparative isoelectric focussing of human apo-HDL, four major forms of apolipoprotein A-I have been isolated. As identified by the following nomenclature and pI, they comprise: apolipoprotein A-I1, pI 5.62; apolipoprotein A-I2, pI 5.53; apolipoprotein A-I3, pI 5.45; apolipoprotein A-I4 pI 5.36. These forms of apolipoprotein A-I were shown to have identical migration on polyacrylamide gel electrophoresis, molecular weights of 26 000 on sodium dodecyl sulfate gel electrophoresis and a common antigenicity with antisera against apolipoprotein A-I or A-I1. Each form had very similar amino acid compositions with the exception of form apolipoprotein A-I4 which contained one isoleucine residue per mol. All forms but apolipoprotein A-I4 were activators of lecithin:cholesterol acyltransferase, the latter was inhibitory to the reaction. From these results, it was concluded that apolipoprotein A-I1, A-I2 and A-I3 are equivalent forms of apolipoprotein A-I whereas apolipoprotein A-I4 is different or heterogeneous. Upon refocussing, the polymorphs were shown to be stable at their pI and not affected by changes in concentration and by the presence of urea or ampholytes. Exposure of a form of apolipoprotein A-I to alkaline pH partially regenerated the original heterogeneity; however, apolipoprotein A-I4 regenerated from apolipoprotein A-I1 did not contain isoleucine, which further demonstrates form apolipoprotein A-I4 heterogeneity.

Studies on the fatty acid binding proteins in cytosol of rat liver.

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.

Purification of human plasma lecithin:cholesterol acyltransferase and its activation by metal ions.

Lecithin:cholesterol acyltransferase (LCAT) has been purified from human plasma by sequential preparative ultracentrifugation, ion-exchange chromatography on DEAE-Sephacel, and affinity chromatography on HDL-Sepharose and on wheat germ agglutinin-Sepharose. After the final step, which included preparative electrophoresis or alternatively, chromatography on hydroxylapatite, a purification of about 24 000-fold was obtained. The LCAT preparation was pure according to alkaline polyacrylamide and SDS-polyacrylamide gel electrophoresis and did not react against antisera to apo AI, AII, and D. The LCAT preparation obtained by preparative electrophoresis was stimulated by Cu2+, Ni2+, Co2+, and Zn2+ at both stages of the reaction, phospholipase reaction and cholesterol esterification. This stimulatory effect was abolished by EDTA.

Heterogeneity of human high density lipoprotein: presence of lipoproteins with and without apoE and their roles as substrates for lecithin:cholesterol acyltransferase reaction.

By affinity chromatography on heparin-Sepharose, two classes of lipoproteins were separated from high density lipoproteins (HDL) isolated from patients with primary or secondary lecithin:cholesterol acyltransferase (LCATase; EC 2.3.1.43) deficiency and from normal subjects. The unretained fraction, HDL(A), was characterized by having apoA-I as a major apoprotein; it also contained apoA-II, -C-II, and -C-III but it contained only traces of immunodetectable apoE and no apoB. The retained fraction, HDL(E), had apoE as the major apoprotein; it also contained apoA-I, -A-II, -B, -C-II, and -C-III. The relative concentration of apoA-I increased with increasing density in the HDL(E) subclass. Compared to HDL(A), HDL(E) had a significantly higher cholesterol content and a lower protein concentration. HDL(E) was mainly (90%) contained within the HDL(2) subfraction. Contamination of HDL(E) by low density lipoproteins (LDL) or Lp(a) was minimal on the basis of pre-beta-electrophoretic mobility and absence of albumin, respectively. Contamination by LDL or Lp(a) could be resolved in part by application of HDL(E) to concanavalin A-Sepharose or to heparin-Sepharose with a shallow gradient. When evaluated as substrates for a highly purified LCATase preparation, the initial reaction rates and V(max) obtained with HDL(A) were always higher than those obtained with HDL(E) in any given plasma. However, both HDL subclasses from LCATase-deficient subjects were better substrates than the corresponding HDL subclasses from normal plasma. Also, both HDL(3A) and HDL(3E) isolated from normal HDL(3) were better substrates than the corresponding subclasses isolated from normal HDL(2). The recognition of this compositional and functional heterogeneity within HDL will allow a better understanding of the metabolism of this lipoprotein class.