Petar Alaupovic

Studies on the composition and structure of plasma lipoproteins: Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins

Abstract To account for the protein heterogeneity of lipoprotein preparations isolated by ultracentrifugal or precipitation techniques, we have proposed a classification system [P. Alaupovic et al., Circulation, 30, Suppl. III (1964) 1; P. Alaupovic, Prog. Biochem. Pharmacol., 4 (1968) 91; P. Alaupovic et al., Expo. Annu. Biochim. Med., 31 (1972)] of plasma lipoproteins based on apolipoproteins as the only distinct chemical components for the differentiation of lipoprotein families. At the present time this concept recognizes the existence of three lipoprotein families, each of which is characterized by the exclusive presence of a single apolipoprotein: lipoprotein family A (LP-A) is characterized by the presence of apolipoprotein A (ApoA), lipoprotein family B (LP-B) by apolipoprotein B (ApoB) and lipoprotein family C (LP-C) by apoliprotein C (ApoC). The qualitative distribution of lipoprotein families in normal human plasma very low density lipoproteins (VLDL, d The LP-A and LP-C families were detected in all lipoprotein density classes; the LP-B family was also present in all classes except the HDL3. Results of the immunological and electrophoretic experiments supported the existence of lipoprotein families as the basic components of plasma lipoprotein system and indicated that lipoprotein families exist above a density range of approximately 1.006-1.019 g/ml as individual separable entities, while at densities less than this range lipoprotein families are present primarily as associations.

Isolation and partial characterization of apolipoprotein D: A new protein moiety of the human plasma lipoprotein system

Immunochemical studies of the major density classes established clearly the antigenic heterogeneity of the human plasma lipoprotein system [ 1 -3 ] . Recent chemical investigations have confirmed these earlier findings and demonstrated the presence of six antigenicalty distinct polypeptides designated as A-I,. A-II, C-I, C-II, C-Ill and LP-B* [4 -6 ] . An additional antigenic component detected predominantly in HDL [7-10] has been designated thin-line polypeptide due to its characteristic appearance on double diffusion analyses of HDL with anti-HDL or anti-whole serum. Under such experimental conditions, the thinline polypeptide appears as a characteristic thin precipitin line nearer the antigen well, while the major precipitin band due to LP-A is closer to the antibody well [9, 11]. The occurrence of thin-line polypeptide has also been demonstrated in LDL [11] and

Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.

Although several authors suggested that the protein moiety of human plasma high density lipoproteins (HDL) (1.063-1.21 g/ m 1) consisted of nonidentical subunits [l-3] , Shore and Shore [4] were first to separate from this protein two polypeptides of similar molecular weight but different amino acid composition and electrophoretic mobilities on polyacrylamide gel. Characterized by threonine and glutamine as C-terminal amino acids, these polypeptides have been referred to as ‘R-Thr’ and ‘R-Glu’, respectively [4] . Although the iv-terminal amino acid analysis of these polypeptides has not yet been reported, previous studies indicated aspartic acid as the sole Ir-terminal amino acid of the intact protein moiety [5]. The occurrence of two nonidentical polypeptides in HDL has been confirmed independently by two groups of investigators [6,7]. We have recently shown [8] that lipoprotein A (LP-A) represents the major lipoprotein family in HDI and that its protein moiety, apolipoprotein A (ApoA), consists of a major and a minor polypeptide characterized by amino acid composition and electrophoretic behavior similar, if not identical, to those of ‘R-‘lhr’ and ‘R-Glu’ polypeptides. In this report we present the results of terminal amino acid analysis of ApoA polypeptides. Contrary to the reports in the literature,

Differential activation of lipoprotein lipase from human post-heparin plasma, milk and adipose tissue by polypeptides of human serum Apolipoprotein C.

Specific polypeptides of human serum apolipoproteins have recently been found to activate triglyceride hydrolysis by LPL* from cow’s milk [l] and rat adipose tissue [2]. These investigators demonstrated a similar polypeptide specificity for activation of the enzyme from both sources. Polypeptides containing glutamic acid or alanine as carboxyterminal amino acid, but not those containing valine, glutamine and threonine, served as activators. The present study was undertaken to evaluate the activation of purified LPL from human postheparin plasma by immunochemically homogeneous polypeptides of human serum APO-A and APO-C and phosphatidylcholine. Inasmuch as the polypeptide specificity of LPL from human post-heparin plasma was found to differ from that reported for LPL from cow’s milk [l] and rat adipose tissue [2] , additional studies were performed using LPL from human milk and adipose tissue as well as purified LPL from dog and rat post-heparin plasma. Purified lipoprotein lipase from post-heparin plasma of normolipidemic subjects was activated

Studies on the interaction of concanavalin A with major density classes of human plasma lipoproteins. Evidence for the specific binding of lipoprotein B in its associated and free forms

Normal human plasma lipoprotein system consists of four distinct lipoprotein families referred to as LP-A,* LP-B, LP-C, and LP-D [l-4]. In the density above 1.019 g/ml, the lipoprotein families occur mainly as separate physicochemical entities [3, 51. However, in the density below 1.019 gJrn1 the lipoprotein families B and C and, if present, lipoprotein families A and D form an association complex(es) [2, 51. In a continuing effort to develop simpler procedures for the isolation and separation of free and associated forms of lipoprotein families, we have

Lipid transport in the avian species Part 1. Isolation and characterization of apolipoproteins and major lipoprotein density classes of male turkey serum

(1) Lipoproteins from the serum of male turkeys maintained on a normal diet were separated by sequential preparative ultracentrifugation into VLDL (d less than 1.006 g/ml), LDL (d = 1.006-1.063 g/ml), HDL (d = 1.063-1.21 g/ml) and VHDL (d greater than 1.21 g/ml). Lipoprotein density classes were characterized by analytical ultracentrifugation, agarose electrophoresis, immunodiffusion and immunoelectrophoresis, and by quantitative determination of protein, lipids and individual phosphatides. (2) HDL were the major density class representing 75% of the total lipoprotein content, LDL accounted for approximately 20% and VLDL for only 3-5% of the total lipoproteins. (3) VLDL were characterized by a relatively low content of glyceride (34%). Cholesterol esters were the major lipid (38%) of LDL, and the phospholipids (26%) of HDL. Glycerides of all major density classes consisted of equal amounts of triglycerides and diglycerides. (4) Phosphatidylcholine was the major phosphatide in all density classes. The composition of phosphatides was very similar in the VLDL and LDL, but it was different in the HDL. The ratio of phosphatidylcholine/sphingomyelin was higher in HDL than in VLDL and LD. (5) Immunological and electrophoretic studies showed that all three major density classes consisted of two lipoprotein families designated, in analogy to the human serum lipoprotein system [1], as LP-A and LP-B. The exception was HDL3 (d = 1.125-1.21 g/ml) which contained only LP-A. (6) ApoB was insoluble in aqueous buffers but could be solubilized after reduction and carboxymethylation. No C- or N-terminal amino acids were released by the usual chemical methods. The carbohydrate moiety of ApoB contained mannose, galactose and galactosamine. (7) ApoA consisted of a non-identical polypeptides designated in analogy to the human polypeptides as A-I and A-II. A-I was the major ApoA polypeptide and had a molecular weight of about 27,000. This polypeptide contained no half cystine, and the aspartic acid as the N-terminal and alanine as the C-terminal amino acids. A-II had a molecular weight of about 10,000, contained no half cystine and had alanine as the C-terminal amino acid. A-II showed no N-terminal amino acid by either dansylation, dinitrophenylation or Edmans procedure. Neither A-I nor A-II contained neutral sugars or hexosamines. (8) Concentrations of polypetides analogous to human ApoC, ApoD and arginine-rich polypeptide, if present, were too low for their unequivocal chemical characterization.

Identification of the protein moiety of an abnormal human plasma low-density lipoprotein in obstructive jaundice

The characteristic elevation of plasma unesterified cholesterol and phospholipid concentrations in sub jects with biliary obstruction is due to the presence of an Abnormal low-density lipoprotein, LP-X, which does not react with antibodies to normal LDL [l-4]. The isolated lipoprotein, LP-X, is characterized by a low content of protein (5%) and a very high content of phospholipid (61-66%) and unesterified cholesterol (23-26%). Since several investigators [5-71 demonstrated immunochemically the presence of LP-A in the LDL fraction, it has been suggested [8,9] that the increased concentration of LDL in subjects with biliary obstruction is caused by a shift of a particular LP-A

Identification of lipoprotein families in familial lecithin: Cholesterol acyltransferase deficiency

Abstract 1. 1. The lipoprotein families in plasma very low density lipoproteins ( d 1 ( d = 1.006−1.019 g/ml), low density Iipoproteins 2 ( d = 1.019−1.063 g/ml), high density lipoproteins ( d = 1.063−1.21 g/ml), and very high density lipoproteins (“1.21 infranate”, d > 1.21 g/ml) isolated from three patients with familial lecithin:cholesterol acyltransferase deficiency were studied by immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. 2. 2. The plasma lipoprotein system in patients with lecithin:cholesterol acyltransferase deficiency was found to contain all the apolipoproteins and their constitutive polypeptides identified previously in plasma lipoproteins of normal subjects. In addition to apolipoprotein B and A-I, A-II, C-I, C-II, and C-III polypeptides, the lipoprotein system in lecithin:cholesterol acyltransferase-deficient plasma also contained an immunochemically distinct apolipoprotein which was previously designated as “thin-line” polypeptide. 3. 3. The qualitative distribution of lipoprotein families in major density classes of the patients plasma closely resembled that of normal, fasting plasma. The lipoprotein B and C families were present as associations in very low density lipoproteins and low density lipoproteins 1 , and as free, unassociated fonns in low density lipoproteins 2 and high density lipoproteins. The lipoprotein A family occurred as free forms in high and very high density lipoproteins. 4. 4. Some major qualitative and quantitative differences were found between the lipoprotein systems of normal and lecithin:cholesterol acyltransferase-deficient subjects. The most significant qualitative difference was the occurrence in the patients plasma of lipoprotein X, the abnormal low density lipoprotein species characteristic of obstructive jaundice. Its concentration in the low density lipoproteins of three patients varied between 49 and 152 mg/1oo ml. The major quantitative difference was the reduced concentration of apolipoproteins A and B in lecithin:cholesterol acyltransferase-deficient plasma. In addition to the previously demonstrated decrease in the concentration of apolipoprotein A (Torsvik H. (1969) Scand. J. Clin. Lab. Invest. 24, 187–196), the concentration of plasma apolipoprotein B was found to be onethird to one-half of that in normal subjects. The low concentration of plasma apolipoprotein B was due to the reduction of apolipoprotein B in low density lipoproteins. The already established differences in the lipid compositions of the major lipoprotein A family from enzyme-deficient and normal subjects were not accompanied by a corresponding change in the polypeptide composition of apolipoprotein A. In both cases, the apolipoprotein A consisted of A-I and A-II polypeptides. The abnormalities of the plasma lipoprotein system in familial lecithin:cholesterol acyltransferase deficiency seem to result from, rather than cause, the virtual absence of enzyme activity.

Utilization of the Quantitative Assay of Lipoprotein X in the Differential Diagnosis of Extrahepatic Obstructive Jaundice and Intrahepatic Diseases

Quantitative determination of LP-X, abnormal serum low density lipoprotein, was performed on the sera of 620 patients with jaundice in two medical centers, one in Oklahoma City, Oklahoma, and the other in Birmingham, England. The results of serial assays over a period of 5 to 8 days after patient admission to hospital or after onset of jaundice, if this occurred in hospital, correlated best with the type and management of jaundice. In some cases of early cholestatic disease of extrahepatic origin LP-X may be absent, but after the observation period it was found that only 1 of 81 (98%) patients with obstruction of the extrahepatic bile duct system remained negative. Of the remainder, 74 (91%) had or developed levels of LP-X exceeding 300 mg per 100 ml. In addition, 43 (88%) of 49 subjects followed serially showed increases in LP-X concentration, with no change in 3 patients. Of 539 subjects with intrahepatic disease, 14 (26.5%) were LP-X positive and 27 (19.4%) of these had initial LP-X levels higher than 300 mg per 100 ml. During the follow-up period, 35 (74%) of 47 patients with intrahepatic disease showed a reduction of LP-X; of the remaining 12 patients 4 had mitochondrial antibody-positive primary biliary cirrhosis, and 6 had severe cholestasis associated with acute infectious hepatitis and high aspartate transaminase levels. Similar figures for alkaline phosphatase showed less consistent changes during the follow-up period. In this retrospective appraisal the trends and absolute levels of LP-X, in addition to the use of similarly followed levels of the routine liver function tests, allowed better differentiation of jaundice requiring surgical correction from that remediable by medical means exclusively than did the use of the routine liver function tests alone. In addition, LP-X is specific for liver dysfunction, whereas other routine liver function tests are not.

Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of C-I polypeptide ("R-Val") of human plasma apolipoprotein C.

Several years ago, results from our laboratory demonstrated [I] that partially delipidized very low density lipoproteins (VLDL, d < 1.006 g/ml) isolated from hypertriglyceridemic plasma contained 3 separable phospholipid-protein residues; the protein moieties of two of these were identical to apolipoproteins A and B (ApoA and ApoB). The protein moiety of the third residue was characterized by a high capacity for phospholipid binding, immunological properties and peptide patterns different from those of ApoA and ApoB, and by serine and threonine as N-terminal amino acids. This newly recognized protein-moiety, designated as apolipoprotein C (ApoC), was also found in the chyle VLDL [2] and in all the major density lipoprotein classes isolated from normal, fakting human plasma [3] . Recently, Brown et al. [4] confirmed the presence in VLDL of 3 phospholipidprotein residues, one of which gave a specific immunoprecipitin reaction only with antibodies to VLDL. In addition, these authors separated the soluble protein moiety of totally delipidized VLDL by gel filtration into 2 major fractions. One fraction consisted of ApoA polypeptides, whereas the second contained a mixture of polypeptides characterized by serine and threonine as N-terminal amino acids. Chromatography of this fraction on a DEAE-cellulose column resulted in the isolation of 3 polypeptides which were designated on the basis of their C-terminal amino acids as apoLP-Val (“R-V