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The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (a-i, a-2, a-3, and jB) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-i) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2).Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freezeetching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles.Glycoproteins comprise about 10% of the total protein of the human erythrocyte membrane (1). Their carbohydrate moieties are antigenic determinants (2, 3) and receptors for viruses and plant agglutinins (4), and their sialic acid residues are responsible for most of the negative charge at the cell surface (5).Glycoproteins have been isolated from erythrocyte membranes with various solvents, such as phenol (6), butanol (7), pyridine (8), sodium dodecyl sulfate (SDS) (9), or formic acid (10 This report summarizes studies on the properties of the major glycoprotein of the human erythrocyte membrane extracted from membranes by a new procedure with lithium di-iodosalicylate as a dissociating agent. A water-soluble glycoprotein has been isolated in high yield that has the chemical properties and biological activities characteristic of the native membrane-bound molecules. This glycoprotein has been partially characterized after tryptic digestion and cyanogen bromide cleavage, and its location in the membrane has been determined by electron microscopy. MATERIALS AND METHODSHuman blood was obtained fresh from donors, and the erythrocytes were washed three times in phosphate-buffered saline (pH 7.4) before preparation of ghost membranes by the procedure of Dodge et al. (11). Glycoprotein was extracted from freeze-dried ghost membranes with 0.3 M lithium diiodosalicylate and purified by phosphocellulose chromatography (12). Samples were electrophoresed in acrylamide gels containing Tris-glycine (pH 8.4), a...

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The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

Many clinical studies have been carried out to determine the health benefits of soy protein and the isoflavones contained in soy. S-equol is not present in soybeans but is produced naturally in the gut of certain individuals, particularly Asians, by the bacterial biotransformation of daidzein, a soy isoflavone. In those intervention studies in which plasma S-equol levels were determined, a concentration of >5-10 ng/mL has been associated with a positive outcome for vasomotor symptoms, osteoporosis (as measured by an increase in bone mineral density), prostate cancer, and the cardiovascular risk biomarkers low-density lipoprotein cholesterol and C-reactive protein. These studies suggest that S-equol may provide therapeutic benefits for a number of medical needs.

Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-I and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II coresponding to residues 1-9 and 10-59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60-78 increased hydrolysis 4fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60-78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55-78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-I. By contrast, the synthetic peptide containing residues 66-78 did not activate. Removal ofthe three COOH-terminal residues, Gly-Glu-Glu, from fragment 60-78 decreased the ability to activate LPL by >95%. These studies suggest that the maximal activation of LPL by apoC-I1 requires a minimal sequence contained within residues 55-78.Chylomicrons and very low density lipoproteins (VLDL) are the vehicles for the transport of plasma triglycerides (1, 2). Transfer of fatty acid from the triglyceride-rich lipoproteins to the tissue requires hydrolysis of the triglyceride by lipoprotein lipase at the capillary walls. Apoliprotein C-II from human VLDL (apoC-II) plays an important role in triglyceride metabolism by serving as an activator of lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3) (3, 4). The amino acid sequence of apoC-I1 (Fig. 1) has recently been determined (5). The protein consists of 78 amino acid residues and is lacking cysteine, cystine, and histidine.We now present results of preliminary studies to define the sequence requirement in apoC-II necessary for the activation of LPL. In addition to testing the three cyanogen bromide (CNBr) fragments corresponding to residues 1-9, 10-59, and 60-78, we have also synthesized and tested the fragments representing residues 66-78, 60-78, and 55-78. On the basis of the results of these studies, we suggest that the sequence determinant in apoC-I1 that is required for maximal activation of LPL is contained between residues 55 and 78. MATERIALS AND METHODSIsolation of apoC-II and CNBr Fragments. apoC-II was isolated as described (6, 7) from VLDL obtained from fasting subjects with type IV or type V hyperlipoproteinemia (8). The isolated apoprotein was homogeneous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate and by amino acid analysis. The three CNBr fragments of apoC-II were prepared by chromatography on Bio-Gel P-30 (5).The peptide corresponding to residues 60-75 was prepared from a tryptic digest of ...