Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins

Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins. The determination of isotopic enrichment of particular amino acids in apolipoproteins is carried out by gas chromatography mass spectrometry (GC-MS). This report describes a robust and sensitive derivative for analysis of d3-leucine by GC-MS and its utility in studying the metabolism of human lipoproteins. The trifluoromethyloxazolinone (oxazolinone) derivative of leucine was formed in a rapid single step procedure using a mixture of trifluoroacetic anhydride (TFAA) and trifluoroacetic acid (TFA). Analysis of the oxazolinone by negative ion chemical ionization GC-MS gave excellent sensitivity and precision, which enabled accurate determination of low levels of isotopic enrichment from small amounts of protein. For example, enrichments between 0.05% and 100% in 100 pg leucine can be measured with a coefficient of variation of <3%. To demonstrate the utility of this procedure, we measured d3-leucine enrichment in apolipoprotein B (apoB) isolated from VLDL and LDL as well as apoA-I isolated from HDL by gel electrophoresis and western blotting. The derivatization procedure gave excellent enrichment data from a single intravenous bolus dose of 5 mg/kg, from which the fractional catabolic rate and production rate of the lipoproteins were calculated. In conclusion, the oxazolinone derivative provides a robust and simple procedure for the sensitive analysis of isotopic enrichment for metabolic studies of human lipoproteins.     

Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics

The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of [5,5,5,-2H3]leucine, [4,4,4,-2H3]valine, and [6,6-2H2,1,2-13C2]lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine, and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.     

Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments

Lipoprotein kinetic parameters are determined from mass spectrometry data after administering mass isotopes of amino acids, which label proteins endogenously. The standard procedure is to model the isotopic content of the labeled precursor amino acid and of proteins of interest as tracer-to-tracee ratio (TTR). It is shown here that even though the administered tracer alters amino acid mass and turnover, apolipoprotein synthesis is unaltered and hence the apolipoprotein system is in a steady state, with the total (labeled plus unlabeled) masses and fluxes remaining constant. The correct model formulation for apolipoprotein kinetics is shown to be in terms of tracer enrichment, not of TTR. The needed mathematical equations are derived. A theoretical error analysis is carried out to calculate the magnitude of error in published results using TTR modeling. It is shown that TTR modeling leads to a consistent underestimation of the fractional synthetic rate. In constant-infusion studies, the bias error percent is shown to equal approximately the plateau enrichment, generally <10%. It is shown that, in bolus studies, the underestimation error can be larger. Thus, for mass isotope studies with endogenous tracers, apolipoproteins are in a steady state and the data should be fitted by modeling enrichments.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Polymorphism of human plasma apolipoprotein C-III at birth and in early neonatal life

Studies were carried out to investigate potential changes in apolipoprotein C-III (apoC-III) polymorphism from birth through the first month of life. Maternal serum at term and neonatal serum at birth, 3, 14, and 28 days were analyzed for the three principal polymorphic forms of apoC-III: apoC-III-0, apoC-III-1, and apoC-III-2. A two-dimensional electrophoretic procedure combining isoelectric focusing and electroimmunoassay was developed for this purpose. In maternal serum (n = 28), apoC-III-1 to apoC-III-2 ratios (1.45) were typical of normolipidemic adult serum, while the corresponding cord sera (n = 32) had a reversal of the apoC-III-1 to apoC-III-2 ratio (0.55). Basic polyacrylamide gel electrophoretic patterns of isolated cord sera lipoprotein density classes confirmed the reversal of the apoC-III-1 to apoC-III-2 ratio in each class. Following birth, the apoC-III-1 to apoC-III-2 ratio reached unity (1.04) at 3 days and did not change significantly thereafter. It can be concluded that the apoC-III-1 to apoC-III-2 ratio in the normal newborn increases shortly after birth primarily due to an increase in apoC-III-1, and this change appears to be associated with oral feeding, suggesting that intestinal factors may play a role in controlling the ratio of plasma apoC-III-1 to apoC-III-2.     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

The distribution and partial characterization of the serum apolipoproteins in the guinea pig.

1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3--4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60--80%) than of LD lipoprotein (40--55%). 6. Small amounts (10--15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10--20%), constituted a major proportion (30--45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.     

Use of Intralipid for kinetic analysis of HDL apoC-III: evidence for a homogeneous kinetic pool of apoC-III in plasma

Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used Intralipid(R) (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 +/- 0.7%) was not different from that of VLDL apoC-III (2.6 +/- 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 +/- 2.9%) (P < 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P < 0.001). In conclusion, this simple IL method can be used to isolate apoC-III from HDL with minimal interference from other HDL apoproteins, and it demonstrates that the kinetics of apoC-III in VLDL and HDL are similar, supporting the concept of a single kinetically homogeneous pool of apoC-III in plasma.     

Lipoprotein apolipoprotein synthesis by human hepatoma cells in culture

Lipoprotein synthesis was demonstrated by double diffusion with low density lipoprotein antibody, and by 3H-labeled amino acid incorporation into proteins of the d less than 1.063 g/ml centrifugally isolated lipoprotein fraction. Radioactive label was incorporated predominantly into apolipoprotein B (60%), apolipoprotein A-I (20%) and apolipoprotein C (12%), as determined by Sepharose column chromatography and polyacrylamide gel electrophoresis. Incorporation of radioactive label into apolipoprotein B was inhibited by the presence of albumin in the medium, and was restored to control levels with the addition of 1 mM oleic acid, indicating that cell synthesis of apolipoproteins could be modified by culture conditions. The human hepatoma cell line, Hep G2, provides a potential in vitro model for the study of regulation of human hepatic lipoprotein and apolipoprotein synthesis.     

Distribution of C apolipoproteins between the vascular and lymph compartments of the rat

This study has investigated the kinetics of transfer of C apolipoproteins between the vascular and lymph compartments of the rat. Very-low-density lipoprotein, labeled with [125I]apolipoprotein C, was injected intravenously into lymph duct-cannulated rats and the redistribution of radioactivity between lymph and plasma followed at frequent intervals for 3 h. Equilibration between the two compartments was rapid (10-15 min), and thereafter removal from both compartments continued at similar rates. Specific radioactivity determinations showed that lymph C-III-0, C-III-3, and C-III-2,1 apolipoproteins rapidly reached values identical to those of corresponding plasma C apolipoproteins and the interrelationship between the curves were consistent with precursor-product relationships in which all, or most, of the product (lymph apolipoprotein C-III) was derived from the precursor (plasma). However, the specific radioactivity curves for C-II peptide did not cross; the lower value for lymph C-II apolipoprotein suggests that, unlike C-III apolipoproteins, a substantial proportion (approx. 40%) of lymph C-II peptide is not derived from the plasma compartment. The most likely source of the unlabeled lymph apolipoprotein C-II is synthesis and secretion from the intestine.     

Fast protein chromatofocusing of human very-low-density lipoproteins

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.     

Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases.

The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.     

Insulin in methionine and homocysteine kinetics in healthy humans: plasma vs. intracellular models

Methionine is a sulfur-containing amino acid that is reversibly converted into homocysteine. Homocysteine is an independent cardiovascular risk factor frequently associated with the insulin resistance syndrome. The effects of insulin on methionine and homocysteine kinetics in vivo are not known. Six middle-aged male volunteers were infused with L-[methyl-2H3,1-13C]methionine before (for 3 h) and after (for 3 additional hours) an euglycemic hyperinsulinemic (150 mU/l) clamp. Steady-state methionine and homocysteine kinetics were determined using either plasma (i.e., those of methionine) or intracellular (i.e., those of plasma homocysteine) enrichments. By use of plasma enrichments, insulin decreased methionine rate of appearance (Ra; both methyl- and carbon Ra) by 25% (P < 0.003 vs. basal) and methionine disposal into proteins by 50% (P < 0.0005), whereas it increased homocysteine clearance by approximately 70% (P < 0.025). With intracellular enrichments, insulin increased all kinetic rates, mainly because homocysteine enrichment decreased by approximately 40% (P < 0.001). In particular, transmethylation increased sixfold (P < 0.02), transsulfuration fourfold (P = 0.01), remethylation eightfold (P < 0.025), and clearance eightfold (P < 0.004). In summary, 1) physiological hyperinsulinemia stimulated homocysteine metabolic clearance irrespective of the model used; and 2) divergent changes in plasma methionine and homocysteine enrichments were observed after hyperinsulinemia, resulting in different changes in methionine and homocysteine kinetics. In conclusion, insulin increases homocysteine clearance in vivo and may thus prevent homocysteine accumulation in body fluids. Use of plasma homocysteine as a surrogate of intracellular methionine enrichment, after acute perturbations such as insulin infusion, needs to be critically reassessed.     

Proteolytic digestion of human serum apolipoproteins by porcine pancreatic elastase

We studied the proteolytic action in vitro of free and alpha 2-macroglobulin-bound porcine pancreatic elastase [EC 3.4.21.11] on the apolipoproteins of plasma: very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion tests of elastase-treated plasma lipoproteins revealed that apolipoprotein C-II and C-III polypeptides were more susceptible to elastase in free form than plasma apolipoproteins (A-I, A-II, B, and E). Elastase bound to alpha 2-macroglobulin did not show any such activities.     

Experimental concerns in tracer kinetic investigations of apolipoprotein metabolism

The complexity of the metabolism of the plasma lipoproteins makes it impossible to integrate the details of the reactions of specific apolipoproteins and their associated lipids without the use of computerized modeling methods. Because apolipoproteins impart specificity in the transport and chemical processing of plasma lipids, they have been the focus of many in vivo kinetic tracer investigations. The analysis of such kinetic data by modeling techniques has provided important advances in understanding lipoprotein metabolism. An example is the Delipidation Chain, an hypothesis explaining VLDL metabolism in terms of a sequential delipidation process. As a consequence of the advance in knowledge of apolipoprotein structure and metabolism, coupled with progress in computerized modeling of large systems, it has become important to refine the design of in vivo tracer kinetic investigations of the apolipoproteins. Considerations of particular importance include the selection of apolipoprotein tracers which can be shown to undergo the same reactions as the apolipoproteins whose metabolism they trace. If the physical and chemical processes which convert apolipoproteins from one metabolic pool to another are to be analyzed correctly, it is necessary to describe precisely and to measure accurately these pools. Current methods for delineating metabolic pools of apolipoproteins in vivo need to be refined. When accomplished, this will provide new opportunities to investigate the metabolic pathways of the apolipoproteins and their associated lipids. A very important challenge is to design experiments which will differentiate transfer processes, which result in net transport of a reactant, from exchange processes, whereby a tracer and a tracee are exchanged between pools without a net transport event occuring. Since both types of processes occur readily with apolipoproteins, it is important to develop methods to examine them separately. Computerized kinetic modeling provides a means for describing and understanding the complexities of lipoprotein metabolism. A major challenge is for the experimentalist to acquire data which accurately reflect the physiological processes involved in lipoprotein metabolism.     

Effects of threonine-poor apolipoproteins on post-heparin plasma hepatic triacylglycerol lipase and lipoprotein lipase

Whole-irradiated rabbit pre-heparin plasma had an important inhibitory effect on hepatic triacylglycerol lipase and lipoprotein lipase activities, whereas control rabbit pre-heparin plasma slightly inhibited hepatic triacylglycerol lipase activity at a high concentration and enhanced lipoprotein lipase activity. As some apolipoproteins were known to modulate these two lipolytic enzymes, the inhibitory effects of irradiated rabbit plasma were investigated in apolipoproteins. Three apolipoproteins, with isoelectric points of about 6.58, 6.44 and 6.12, characterized by their low content in threonine (threonine-poor apolipoproteins) were produced in high concentrations in rabbit VLDL and HDL after irradiation. The effects of these apolipoproteins on control rabbit post-heparin plasma hepatic triacylglycerol lipase and extrahepatic lipoprotein lipase were studied. Threonine-poor apolipoproteins substantially inhibited the hepatic triacylglycerol lipase activity and enhanced the apolipoprotein C-II-stimulated activity of lipoprotein lipase. The amounts of these apolipoproteins in triacylglycerol-rich lipoprotein particles may determine the lipolytic activity of lipoprotein lipase and hepatic triacylglycerol lipase in triacylglycerol hydrolysis. The existence of another inhibitor of lipoprotein lipase remains to be determined.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

cDNA sequences of two apolipoproteins from lamprey

The messages for two small but abundant apolipoproteins found in lamprey blood plasma were cloned with the aid of oligonucleotide probes based on amino-terminal sequences. In both cases, numerous clones were identified in a lamprey liver cDNA library, consistent with the great abundance of these proteins in lamprey blood. One of the cDNAs (LAL1) has a coding region of 105 amino acids that corresponds to a 21-residue signal peptide, a putative 8-residue propeptide, and the 76-residue mature protein found in blood. The other cDNA (LAL2) codes for a total of 191 residues, the first 23 of which constitute a signal peptide. The two proteins, which occur in the "high-density lipoprotein fraction" of ultracentrifuged plasma, have amino acid compositions similar to those of apolipoproteins found in mammalian blood; computer analysis indicates that the sequences are largely helix-permissive. When the sequences were searched against an amino acid sequence data base, rat apolipoprotein IV was the best matching candidate in both cases. Although a reasonable alignment can be made with that sequence and LAL1, definitive assignment of the two lamprey proteins to typical mammalian classes cannot be made at this point.     

Isolation, characterization and comparative aspects of the major serum apolipoproteins, B-100 and AI, in the common marmoset, Callithrix jacchus

The two major apolipoproteins of marmoset serum have been isolated and characterized, and on the basis of physicochemical and immunological criteria are homologous with the human AI and B-100 proteins. Marmoset apolipoprotein AI was the principal protein of high-density lipoproteins (HDL) and was purified by gel filtration chromatography and electrophoresis in alkaline-urea polyacrylamide gel followed by electrophoretic elution. Purified marmoset apolipoprotein AI displayed an Mr of approx. 27000, was polymorphic (five forms) on isoelectric focussing, with pI values in the range 4.8-5.0, and migrated similarly to human apolipoprotein AI in alkaline-urea gels. An overall resemblance was seen in the amino acid composition of marmoset apolipoprotein AI and that of its human counterpart with the notable exception that marmoset AI contained 1 isoleucine residue/mole. An immunological reaction of partial identity between the human and monkey proteins was seen upon immunodiffusion of their HDLs against antiserum to human apolipoprotein AI. Marmoset B-100 was the predominant apoprotein of VLDL and LDL, resembling the human protein in its elution profile on gel filtration chromatography in anionic detergent, and in its high apparent Mr (approx. 520000). The marmoset and human B-100 proteins were alike in amino acid composition and carbohydrate content. Moreover, their immunological behaviour with an antiserum to marmoset apolipoprotein B showed them to share certain antigenic determinant(s). We conclude that the physicochemical properties of the principle apolipoproteins of Callithrix jacchus, a New World primate, markedly resemble those of the human AI and B-100 proteins, suggesting therefore that they may function similarly in lipid transport and metabolism. Counterparts to human apolipoproteins AII, E, CII and CIII have also been tentatively identified.