The metabolism of very low density lipoprotein proteins

BIOCHIMICA ET BIOPHYSICA ACTA zyxwvutsrqp 94 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA BBA $097 THE II. METABOLISM STUDIES OF ON THE VERY LOW TRANSFER DENSITY LIPOPROTEIN OF APOPROTEINS PROTEINS BETWEEN PLASMA LIPOPROTEINS SHLOMO EISENBERG*, DAVID W. BILHEIMER** I. LEVY*** zyxwvutsrqponmlkjihg ROBERT AND Section on Lipoproteins, Molecular Disease Branch, National Institutes of Health, Bethesda, &Id. aoor~ (U.S.A.) Heart and Lztng Institute, National March n4th, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1972) (Received SUMMARY I. Apolipoprotein-glutamic (apoLP-Ala), small molecular very low density protein lipoprotein always exceeds concentration radioactivity density at least 2. that to low density Apolipoproteins predominantly Apolipoprotein-glutamine, reassociate primarily proteins glyceride). and involve (apoLP-Gln,) with their parent of both groups The recombination a process and is proportional of apoLP-Glu In contrast, of apoproteins be- and thus rethe apoprotein in this type of transfer. their reassociation reassociate and high density and apolipoprotein-glutamine, lipoprotein, of and high and apoLP-Ala is bidirectional, and apoLP-Ala very low density lipoto the A similar transfer into groups following ApoLP-Glu however, of “ recognition” to high density mixture. phenomenon. can be separated in vitro from transfer to both plasma triglyceride does not participate with lipids and lipoproteins. all plasma lipoproteins, sentative lipoproteins, lipoproteins an exchange lipoprotein apolipoprotein-alanine Their transfer levels. The transfer and high density in part, and readily present in the incubation cholesterol of low density properties to other lipoproteins. of lipoproteins lipoprotein presents, (apoLP-Glu) occurs in viva., and is proportional tween very low density moiety acid weight apolipoproteins, high density reassociate with lipoproteins of the lipoprotein (apoLP-Gln,) lipoprotein. with lipid with lipoprotein. (lecithin Repreor tri- thus may be specific by the apoprotein. This Abbreviations: apoLP-Ser, apolipoprotein-serine; apoLP-Glu, apolipoprotein-glutamic acid; apoLP-Ala, apolipoprotein-alanine; apoLP-Gin,, apolipoprotein-glutamine, (see footnote on p. 95 : apoLDL, apoprotein moiety of low density lipoprotein; apoLP-Gin,, apolipoprotein-glutaminee; apoVLDL, apoprotein moiety of very low density lipoprotein; apoHDL, apoprotein moiety of high density lipoprotein. * On leave from the lipid research laboratory, Department of Medicine B, Hadassah University Hospital, Jerusalem, Israel. ** Section on Lipoproteins, Molecular Disease Branch, National Heart and Lung Institute. Bethesda, Maryland. * * * Address all request for reprints to Robert I. Levy, Molecular Disease Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md. 20014, U.S.A. Biochim. Biophys. 24cta, 280 (1972) g+Iod APOPROTEIN TRANSFER BETWEEN LIPOPROTEINS 95 specificity may not be involved in the simple recombination of apolipoproteins and lipids. These observations may explain the distribution of apoproteins among plasma lipoproteins and provide insight into their metabolic fate. INTRODUCTION Recent studies of the protein portion of the lipoproteins have shown that each lipoprotein family contains a spectrum of apoproteinsl-s. Most of these apoproteins, moreover, are present in more than one lipoprotein family”ya. One apolipoprotein, designated apoLDL, is present in both very low density and low density lipoproteins. A group of small molecular weight proteins (apoLP-Ser, apoLP-Glu and two species of apoLP-Alaa-5r7) constitute about 50% of apoVLDL, 5-15 o/o of apoHDL2-6, and are present in trace amounts in low density lipoproteins. Two other apolipoproteins which are the major high density lipoprotein apoproteins8g10,(apoLP-Gin,)” and apoLPGin, may be present in very low density lipoprotein ll. In addition, other proteins present in small amounts in very low density or high density lipoproteinsa- may be common to more than one lipoprotein family. Though these data suggest some relationship between similar apoproteins on different lipoproteins, the nature of this relationship has been unclear. In a previous reportI we demonstrated that following a short in zyxwvutsrqponmlkjih vitro incubation of 1251-labelledvery low density lipoprotein with plasma, apoLP-Glu and apoLPAla,+, readily transferred from very low density lipoprotein to other lipoproteins, mainly high density lipoprotein. The present study extends these observations and includes experiments on the in vitro behavior of isolated, purified apolipoproteins. MATERIALS AND METHODS Preparation of radioiodi?tated lipoproteins Lipoproteins were isolated from fresh plasma (obtained from normal fasting humans or when indicated, from patients with hyperlipoproteinemia) in 0.1 y. EDTA by differential centrifugation using the Beckman Model L2-65B ultracentrifuge as previously described 12.By immunodiffusion techniques and immunoelectrophoresislS very low density and low density lipoproteins were shown to be free of other contaminant plasma proteins and lipoproteins; some preparations of high density lipoprotein contained minimal amounts of low density lipoprotein (estimated as less then 3 %). Very low density lipoprotein was radiolabelled with I261using a slight modification12 of the ICL iodination method of MacFarlane I*. Iodination of high density lipoprotein was performed following methods essentially identical to those used for labelling very low density lipoprotein. The efficiency of labelling of high density lipoprotein was as high as 71 o/O,and the amount of la61bound to lipids did not exceed 1.2 o/O.The ratio of atoms of iodine per mole of high density lipoprotein apoprotein was 0.25, assuming a molecular weight of 25000 or less for the apoproteins?. l Until recently the carboxyl terminal amino acid of this apoprotein was thought to be threoninee. Revent evidencelo indicates that the carboxyl terminous is glutamine and we will use the terminology apoLP-glutamine, and apoLP-glutamine, to refer to the major high density lipoprotein apoproteins. Biochim. Biophys. 4cta, 280 (1972) g+roq S. EISENBERG 96 Radioactivity was determined using Packard 5ozz or 3375. Counts in the different quenching carrier due to high salt concentration free was purchased Prior l351-labelled injection very low density into lipoprotein pm) and shown to be free of pyrogens Imubation otherwise indicated, for 30 min in 5o-ml Erlenmeyer tion, the samples were placed cellulose nitrate incubated directly Boston, standard. Mass., U.S.A. or hyperlipoproteinemic was sterilized by millipore and bacteriological for NaW patients, filtration (0.45 contamination15. all ilz vitro incubations on crushed ice, and 5-ml aliquots tubes. In some experiments, in 6.5-ml were carried out at 37 “ C flasks, air being the gas phase. At the end of incuba- cellulose nitrate were transferred 5-ml mixtures ultracentrifuge were prepared tubes. to and The density was to either zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB 1.006, 1.019 or 1.21 with NaCl-KBr solutions of known densi- then adjusted ties or solid KBr, 40000 Nuclear, normal Nos. were corrected jvocedwes Unless 6.5ml spectrometers, subfractions by using Na lz61 as internal from New England to intravenous autogamma ultracentrifugal zyxwvutsrqponmlk et al. rev./ min and the samples were spun in a Beckman (IOO ooo x g). Following ultracentrifugation, in the middle of the clear zone, and the resultant 40.3 rotor for 24 h at the tubes tops and bottoms were sliced were taken for assay of radioactivity. To investigate were prepared acid poor bovine density the interaction by I min sonication albumin in 0.85% of each incubation mixture lipids were recovered at 40000 rev./ min Delipidation, of labelled apoproteins of lecithin in 0.85% NaCl. Following was adjusted with lipid, lipid emulsions NaCl or triglyceride 30 min incubations in I Th fatty at 37 “ C, the to 1.21 with NaCl and KBr, in the top 2 cm of the tube after centrifugation and the in a 40.3 rotor for 24 h. gel filtration, ion exchange chromatography and polyacrylamide gel electvo- phoresis Delipidation, gel filtration, gel electrophoresis termine the distribution gels, stained assayed of radioactivity with 0.05% chromatography procedures previously and polyacrylamide described3-6t12. among apolipoproteins, Very low density lipoproteins, and the slices were were sliced from the poly- described 12. Low and high density gels as previously To de- 10% polyacrylamide bluele, were sliced by hand Coomassie for radioactivity. acrylamide ion-exchange were done following lipoprotein apopro- teins were sliced in a similar manner. To determine radioactivity tube were transferred plot the radioactivity Sephadex proteins columns. emerged chromatography order to obtain in the column to Packard counting fractions, tubes and curves. These curves corresponded On DEAE ion exchange after the protein to combine peaks. chromatography, fraction, from each counts with the protein however, It was thus found necessary tubes from the descending a pure apoprotein 50-~1 aliquots the resultant the labelled after DEAE limb of each protein uncontaminated used to peaks of the peak in by radioactivity from other proteins. Isolation of -I-labelled Labelled low density Biochim. apoproteins apoproteins or high density Biophys. Acta, and apoprotein fractions were obtained from human very lipoproteins. Following delipidationa, apoproteins 280 (1972) 94-104 were APOPROTEIN TRANSFER BETW EEN LIPOPROTEINS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO 97 zyxwvutsrq solubilized in 0.2 M Tris buffer (pH 8.2), 6 M urea and to 2-5 mg protein. No detergent was used. 0.01% EDTA, using I ml buffer of high density lipoprotein origin The delipidated radioactive high density lipoprotein apoproteins were separated by gel filtration on Sephadex G-200 into three major peaks. The first and second peaks were identified on polyacrylamide gel electrophoresis to correspond to the two major high density lipoprotein apoproteins, apoLP-Gln, and apoLP-Gln, The third peak consisting of 5 bands on polyacrylamide gels was further resolved on DEAE ionexchange chromatography to yield four radioactive protein fractions. The first fraction emerged at conductivity of 1.5 mCI and consisted of two adjacent protein bands on polyacrylamide gel with an RF intermediate between that of apoLP-Gln, and apoLP-Glu. This fraction is designated HD-I The second, third and fourth fractions corresponded to apoLP-Glu, apoLP-Ala, and apoLP-Ala, as judged immunochemically7. The two alanine species were combined and subsequently used together. Apoproteins of very low density lipoprotein origin About 50% of the radioactive delipidated very low density lipoprotein apoproteins were solubilized in the Tris-urea buffer. By gel filtration on Sephadex G-150 followed by DEAE ion exchange column chromatography, the following labelled fractions and apoproteins were isolated: Sephadex Fraction II (VSF-II) consisting of a single protein band on polyacrylamide, which had a RF similar, but not identical, to that of apoLP-Gin,. Sephadex Fraction III (VSF-III) contained four apoproteins, apoLP-Ser, apoLP-Glu and apoLP-Ala,+,. Purified preparations of labelled apoLPGlu, apoLP-Ala, and apoLP-Ala, were obtained from the DEAE column. ApoLPSer contained a minimal amount of radioactivity probably owing to the absence of tyrosine in this proteina. Its behavior could therefore not be followed by tracer technique. The apoLDL moiety of very low density lipoprotein apoproteins did not dissolve in the Tris-urea buffer which did not contain any detergent and was separated as an amorphous precipitate from the apoprotein solution prior to gel filtration3. All apoproteins and apoprotein fractions were dialized against water-0.1% EDTA (pH 8.2) and concentrated on diaflo membranes. In each case, the stained band (or bands) sliced from polyacrylamide gel contained 85-957, of the total gel radioactivity. Apoproteins RESULTS using labelled lipoproteins The in vivo transfer of autologous W-labelled very low density lipoprotein radioactivity to other lipoproteins has now been recorded in 13 normal subjects or patients with various types of hyperlipoproteinemia IO min after intravenous injection of labelled very low density lipoprotein* (Table I). In the Io-min sample radioactivity originally present in very low density lipoprotein was recovered as well as in Studies * A detailed report of the in viva metabolism of the very low density lipoprotein apoproteins in these patients studied over a rc+rq-day period is the subject of another communication. It has been reported in preliminary form”. Baochim. Biophys. Acta, 280 (1972) 94-10~ S. EISENBERG et zyxwvutsrqpon al. 98 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE I DISTRIBUTIONOF RADIOACTIVITY zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB AMONG PLASMA LIPOPROTEINS IO MIN AFTER INJECTION OF lzaILABELLED VERY Subject LOW DENSITY LIPOPROTEIN TO HUMANS Triglycerides zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Cholesterol (mg/Ioo ml) Distvibution of radioactivity ilt lipoproteins ( y0 of total) (mg/Ioo ml) Density lipoprotein LOW High Normal Normal Type IV Type III Type III Normal 65 90 116 136 14’ 149 38 40 40 69 40 Type Type Type Type Type Type Type 290 296 1-i 34 34 32 36 25 23 V III III IV III IV IV 306 312 450 jI0 544 I’7 90 104 62 46 43 26 22 IO1 93 I’7 152 73 83 Density lipoprotein Very low d < 1.006 594 72.8 73.4 59.5 77.6 So.6 86.2 84.6 80.0 80.0 80.5 88.7 88.2 LOW d 1.019-1.063 High d 1.063-1.21 0.9 3.8 23.6 19.7 6.1 12.5 6.9 3.4 q.2 2.2 10.6 4.3 I.4 I.9 4.3 2.4 6.7 7.9 10.3 7.0 5.3 6.6 5.6 0.I I.1 13.5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA RATIO OF '=I,VLDL/HDL Fig. 1. Distribution of radioactivity between very low density lipoprotein (VLDL) and high density lipoprotein (HDL) (abscissa) as plotted against plasma triglyceride levels (ordinate). Values recorded IO min after injection of 1251-labelled very low density lipoprotein to 14 humans. high density lipoprotein tween very low density triglyceride (d 1.063-1.21) (Table I). The distribution and high density lipoproteins was found of radioactivity to parallel be- plasma levels (Fig. I). In vitro transfer In order to further define this transfer of radioactivity between the lipoprotein species isolated purified lipoproteins were incubated in v&o. Only trace amounts of radioactivity were recovered in the subfraction of density greater than 1.019 when l*SI-labelled very low density lipoprotein was incubated with 0.85 NaCl solution or with plasma proteinsof density greater than 1.21. However, after the addition of either low density or high density lipoproteins to labelled very low density lipoprotein, appreciable amounts of protein-bound radioactivity appeared in the subfraction of Biochim. Biophys. Acta, 280 (1972) 94-104 99 APOPROTEIN TRANSFER BETWEEN LIPOPROTEINS TABLE II VERY LOW DENSITY LIPOPROIn zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vit r o zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TRANSFER OF LABELLED APOPROTEINS FROM “61-LABELLED TEIN TO LOW DENSITY LIPOPROTEIN OR HIGH DENSITY LIPOPROTEIN Conditions of incubation : As described in Materials and Methods. Lipoproteins were separated at d r.org and the distribution of radioactivity between the top 2 cm of the tube (very low density lipoprotein) and bottom of tube (low density lipoprotein or high density lipoprotein) was determined. The distribution of labelled apoproteins in very low density lipoprotein before incubation and low density lipoprotein or high density lipoprotein after incubation is shown in Table 3. zyxwvutsrqponm Incubation mixture lssI-labelled very low Low 07 high density lipoprotein density lipoprotein (mg protein) (mg protein) Radioactivity transfevred to low density lipoprotein 07 high density lipopuotein ( yO of total) Incubation with low density lipoprotein Exp Exp I 0.5 0.5 0.5 0.5 0.5 TABLE 1.0 5.7 2.5 5.0 10.0 Expt 2 13.2 4.9 8.0 12.5 ::: 13.1 18.3 8.5 12.3 17.0 18.6 15.3 25.2 22.2 III DENSITY Expt* Expt I 2 I.5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM 2.8 2.8 - RADIOACTIVE LOW Incubation with hieh density lipop70teinv APOPROTEINS LIPOPROTEIN Lipoprotein IN LIPOPROTEINS OR ‘*51-LABELLED BEFORE HIGH AND AFTER DENSITY INCUBATION WITH “‘I-LABELLED VERY LIPOPROTEIN Distribution of lasI-labelled apoproteins (Oh of total) Zone r (apoLDL) Zone 3 zone 5 (apoLP-Glu) Zone 6 (apoLP-Ala) Recovery (%) Very low density lipoprotein 8.6 46.3 3.7 34.6 93.2 Low density lipoprotein, after incubation with I 12.5 87.2 3.8 63.5 7.4 High density lipoprotein, after incubation with I 64.6 87.0 3.8 9.3 9.3 Very low density lipoprotein 88.7 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA I 45.6 5.4 4.1 33.3 Low density lipoprotein, after incubation with ,I 12.3 8.5 63.5 91.4 7.’ II High density lipoprotein, after incubation with I 66.0 87.8 7.2 7.1 7.5 Very low density lipoprotein, after incubation with II 16.6 2.3 4.5 72.5 95.9 I I Distribution of radioactivity among apoproteins was determined by counting protein bands sliced from polyacrylamide gels. * Numbers refer to experiments described in Tables II en V. density greater than I.019 (Table II). At all lipoprotein-protein ratios, the transfer of radioactivity to high density lipoprotein exceeded that transferred to low density lipoprotein. This transfer of radioactivity was proportional to the lipoprotein-protein ratio in the incubation mixture (Table II). In agreement with our previous resultsla, the labelled proteins that transferred were mainly apoLP-Glu and apoLP-Ala,+, (Table III). Radioactive lipids also transferred from’ very low density lipoprotein to low density or high density lipoprotein in these experiments. The ratio of transferred lipids to transferred proteins was similar at all lipoprotein concentrations averaging 23% for low density lipoprotein and 14% for high density lipoprotein. When very low density lipoprotein was incubated with a mixture of low density and high density lipoproteins, the amount of radioactivity recovered in the high density lipoprotein fraction exceeded that in low density lipoprotein again demonBiochim. Wophys. Acta, 280 (1972) g4-ro4 100 TABLE S. EISENBERG et al. IV 1% zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA V ih’O TRANSFER OFLABELLED APOPROTEINS FROM Iz51-LA~E~~~~ VERY LOW DENSITY LIPOPROTEINTOLOWDENSITYLIPOPROTEINAND HIGHDENSITYLIPOPROTEIN Conditions of incubation as described in Materials and Methods. Lipoproteins were separated at d I.019 and 1.063 to obtain the distribution of radioactivity in lipoproteins of d ~.o~g-1.063 (low density lipoprotein) and d > 1.063 (high density lipoprotein). zyxwvutsrqponmlkjihgfedc zyxwvutsr zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ..___ Incubation mixture of radioactivity (O,] of total) Distribution ‘251~labelled very low density lipoprotein (mg protein) Low density lipoprotein High density lipoprotein d I.OIg-I.063 d > 1.063 (mg protein) (mg protein) kxpt I Expt I 0.5 - - j 5 0.5 I 0.5 5 5 I 0 .j TABLE Expt 2 0.4 0 6 .5 7 .9 5.2 I I .0 2 .4 ‘3.7 ‘4.7 3.0 I O.1 6 .9 Expt 2 1 ..j rj.0 1 7 .0 6.7 V TRANSFEROFLABELLED LIPOPROTEIN APOLP-CLUAND APOLP-.~LAFROMHIGHDENSITYTO"ERYLOWDENSITY Conditions of incubation as described in Materials and Methods. Lipoproteins were separated at d 1.019 to obtain top 2 cm of tube (very low density lipoprotein) and bottom of tube (high density lipoprotein). Incubation mixture very low density lipoprotein (mg pvotein) 1e51-labelled high density lipoprotein (mg protein) Radioactivity in very low density lipoprotein (“/L of total) Expt I Expt 2 0.6 76.9 83.9 89.0 0 I 18.9 I 2.5 I I 66.2 68.2 x5.7 IO I ._ * High density lipoprotein, labelled predominantly in apoLP-Glu and apoLP-Ala was prepared by incubation of high density lipoprotein with iz51-very low density lipoprotein. The distribution of labelled apoproteins in the high density lipoprotein is shown in Table III (high density lipoprotein, after incubation with I). Also, shown in Table III are the labelled apoproteins of the very low density lipoprotein, after incubation with the iz51-labelled high density lipoprotein (very low density lipoprotein, after incubation with II). strating- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA that the transfer of apoLP-Glu and apoLP-Ala from very low density lipolipoprotein exceeds that to low density lipoprotein (Table IV). The bi-directional mode of transfer of apoLP-Glu and apoLP-Ala is shown in Table V. In this experiment, high density lipoprotein was labelled by preincubation with very low density lipoprotein and then isolated and reincubated with unlabelled very low density lipoprotein (apoLP-Glu and apoLP-Ala constituted more than 709& of the high density lipoprotein radioactive proteins). More than 80% of the radioactivity introduced into the high density lipoprotein subfraction was transferred back to very low density lipoprotein (Table V). protein to high density Eqberiments with isolated apoproteins Following incubation with plasma, two different apoprotein interactions were observed. VSF-II, VSF-III (defined as described in the Methods Section), apoLP-Glu and the two apoLP-Ala species from either very low density or high density lipoBiochim. Biophys. Acta, 280 (1 9 7 2 ) 9 1 -1 0 4 APOPROTEIN TABLE VI BETWEEN IO1 LIPOPROTEINS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA DISTRIBUTION DENSITY TRANSFER OF “ 51-LABELLED LIPOPROTEIN ORIGIN VERY AMONG LOW DENSITY PLASMA LIPOPROTEIN LIPOPROTEINS lp61-LABELLED AND APOPROTEINS OF VERY LOW zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON Conditions of incubation as described in Materials and Methods. Labelled very low density lipoprotein or apoproteins were incubated with human plasma. Plasma lipids: Expt 1: 144 mg/1oo ml triglycerides, 31 mg/Ioo ml high density lipoprotein cholesterol and 131 mg/roo ml low density lipoproteincholesterol. Expt 2 :44 mg/Ioo ml triglycerides, 54 mg/Ioo ml high density lipoprotein cholesterol and 113 mg/1oo ml low density lipoprotein cholesterol. Incubation mixture Labelled compound pg protein Distribution d < 1.006 Expt I Very low density lipoprotein VSF-II VSF-III apoLP-Glu apoLP-Ala, apoLP-Ala, 138 155 160 55 56 55 80.5 23.2 54.4 59.0 51.0 47.8 Expt 2 Very low density lipoprotein VSF-II VSF-III apoLP-Glu apoLP-Ala, 138 155 160 55 55 67.7 12.0 24.6 32.1 23.8 of radioactivity d 1.006-1.019 in lipoproteins ( yO of total) d 1.019-1.063 d 1.063-1.21 2.3 5.9 8.2 5.7 6.4 6.7 8.5 10.5 15.1 17.8 7.6 35.4 23.1 17.3 24.6 22.5 2.9 2.8 5.7 6.7 3.6 8.6 8.2 19.2 13.0 21.1 16.4 46.6 40.6 45.4 49.2 12.9 II.2 d > 1.21 3.9 29.1 1.4 4.6 30.4 9.9 2.8 2.3 2.0 2.6 3.4 * The very low density lipoprotein contained 10.1 o/o of the *as1attached to lipids. The distribution of isa1 among proteins was 49.9% bound to apoLDL, 5.5 % to proteins of Zone 3, 13.3% to apoLP-Glu and 27.3% to apoLPAla,+,. TABLE VII DISTRIBUTIONSOF lasI-~~~~~~~ APOPROTEINSISOLATEDFROM PLASMA l*sI-~~~~~~~~ HIGH DENSITY LIPOPROTEIN AMONG LIPOPROTEINS Conditions of incubation as described in Materials and Methods. Labelled apoproteins were incubated with human plasma. Incubation mixture Labelled compound apoLP-Gin, apoLP-Gin, HD-I * apoLP-Ala,+, pg protein I: 63: Distribution of radioactivity d < 1.006 d 1.006-1.019 (% of total) d r.oIg-I.063 d 1.063-1.21 d > 1.21 0.3 0.3 3.0 40.3 0 0.1 1.0 0.8 0.5 9.6 92.0 :::: 6.7 3.0 13.7 6.1 s.5 37.5 * This apoprotein fraction is described in Materials and Methods. proteins (Tables VI and VII), distributed among all the plasma lipoproteins, mainly very low density and high density lipoproteins. The distribution of the apoproteins was dependent, at least in part, on the initial ratio of lipoproteins in the plasma (Table VI). In contrast, apoLP-Gin,, apoLP-Gln, and the proteins in the HD-I fraction, reassociated only with their parent lipoprotein, e.g. high density lipoprotein (Table VII). ApoLP-Ala, and apoLP-Gln, were chosen as representative proteins for a more detailed study of differential lipid and lipoprotein interaction. Marked dissimilarity was found between the behavior of these two proteins. More than 95 y0 of the apoLPAla, protein reassociated with very low density, low density, or high density lipoprotein (Table VIII). Only a small amount of apoLP-Gin, was found to associate with very low density or low density lipoprotein, even when they were the only lipoproteins Biochim. Biophys. Acta, 280 (1972) 94-104 102 TABLE S. EISENBERG et al. zyxwvutsrqp VIII zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA REASSOCIATION OF APOLP-ALAe AND APOLP-GLN, WITH LIPOPROTEINS Conditions of incubation: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB zopg of labelled apoproteins were incubated with lipoproteins as described in Materials and Methods. Libobrotein 1 1 mixture Very low den&v lipo>rotein (mg protein) * Radioactivity Low densitv lipoprotein* (mg protein) Hieh densitv lip&otein d (mg protein) in top zcm of tube (% of total) * ApoLP-Ala, d < I.OI9 ApoLP- Gin, d < 0 0.5 0 0 0 5 0 0 96.6 5 5 0 93.9 98.6 0 0.5 0.5 0 1.019 d < 1.21 5 5 96.2 IO..+ 65.1 71.0 31.6 78.7 0.9 1.5** 5 * Incubation mixtures separated to top 2 cm of tube and bottom of tube as densities of r.org or to determine the distribution of radioactivity following centrifugation. ** Lipoproteins separated at density of 1.063. TABLE zyxwvutsrq zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1 1 .0 13.3 3.1 15.0 0 0 d < I.ZI 1.~ 1 IX REASSOCIATION OF APOLP-ALAs AND APOLP-GLN1 WITH Conditions of incubation : 20 pg of labelled apoproteins as described in Materials and Methods. ARTIFICIAL LIPID were incubated EMULISONS with 5 ml lipid emulsions, Radioactivity in top z cm of tube ( y 0 of total) ** Lipid* ApoLP-Ala, None Lecithin, 0.1 mg/ml Lecithin, I mg/ml Triglyceride, o. r mg/ml ApoLP- Gin, IO.5 12.9 86.5 96.4 78.3 95.9 98.5 60.5 * Lipid emuslsions were prepared as described in Materials and Methods. ** The incubation mixtures were separated to top L cm (containing the lipids) and bottom centrifugation at density of I. 2I. by in the incubation system (Table VIII). ApoLP-Ala, distributed between very low density and low density or high density lipoprotein when mixtures of lipoproteins were used though its affinity for very low density lipoprotein was greatest; under similar conditions, apoLP-Gin, did not associate in appreciable amounts with either very low density or low density lipoprotein (Table VIII). In contrast, when the interaction of apoLP-Ala, and apoLP-Gln, was investigated using lecithin or triglyceride emulsions, the bulk of both proteins was found in association with the lipids, in the d 1.21 supernatant. DISCUSSION We have recently shown that the fate of labelled apoLP-Glu and apoLP-Ala in very low density lipoprotein differs from that of apoLDL12. In zyxwvutsrqponmlkjihgfedcbaZY vitro, apoLP-Glu and apoLP-_4la are characteristically transferred from very low density lipoprotein to other plasma lipoproteins whereas apoLDL is not involved in this transfer. In vivo, apoLDL disappears with time from the very low density lipoprotein density range faster than apoLP-Glu and apoLP-Alala. Biochim. Biophys. Acta, 280 (1972) 94- 104 APOPROTEIN TRANSFER BETWEEN LIPOPROTEINS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP 103 zyxwvutsrq The experiments described herein confirm and extend our initial observations and show that under a variety of conditions labelled apoLP-Glu and apoLP-Ala are transferred from very low density lipoprotein to other lipoproteins,mainly high density lipoprotein. Under these conditions, the apoLDL portion of very low density lipoprotein is not transferred (Table III). Since apoLP-Glu and apoLP-Ala also readily transfer back to very low density lipoprotein from high density lipoprotein (Table III), we propose that this movement of apoproteins between lipoproteins represents, at least in part, an exchange phenomenon similar to that described for phospholipids17 and free cholesterolls. This exchange may preserve apoLP-Glu and apoLP-Ala in the plasma during very low density lipoprotein catabolism, and explain our previous observation that following injection of W-labelled very low density lipoprotein into humans, the apoLDL portion of very low density lipoprotein disappears rapidly from very low density lipoprotein (presumably reflecting catabolism of very low density lipoprotein molecules) whereas apoLP-Glu and apoLP-Ala decay at a much slower rate12. The distribution of apoLP-Glu and apoLP-Ala between very low density lipoprotein and low density or high density lipoprotein in vitro was proportional to the concentration of lipoproteins in the plasma or incubation mixtures. In vivo, this distribution was primarily determined by the plasma triglyceride levels (and presumably therefore the total very low density lipoprotein concentration). However, at any triglyceride concentration, this distribution was also influenced by the ratio of plasma very low density to high density lipoprotein concentrations (Table I). Thus, one may speculate that diet or drugs, which are known to alter the levels of plasma very low density or high density lipoproteinslo may also cause a change in the distribution of apoLP-Glu and apoLP-Ala between lipoprotein families. ApoLP-Glu and apoLP-Ala reassociate with all plasma lipoproteins; apoLPGln, and apoLP-Gln, reassociate only with high density lipoprotein. Only avery small amount of apoprotein is recovered with the remainder of the plasma proteins (d > 1,s~). Similar results were previously reported when the association of a mixture of apoproteins obtained from high density lipoprotein with plasma lipoproteins was examinedzo-22. In contrast, 1251-labelled albumin or W-labelled y-globulin did not associate with lipoproteins and most of their radioactivity was recovered with the plasma protein fraction of density greater than 1.21ao+. It has also previously been reported that a substantial amount of labelled high density apoprotein reassociated with either chylomicrons or low density lipoprotein when high density lipoprotein was absent from the incubation mixture 30-21.Since the several apoproteins present in high density lipoprotein differ in their affinity towards lipoproteins (Table VII), it is impossible to conclude from these older studies whether all or only several of the high density lipoprotein apoproteins reassociated with lipoproteins other than high density lipoprotein. Although a substantial amount of both apoLP-Ala and apoLP-Gln, were found to reassociate with lipids (lecithin or triglyceride), their afhnity towards lipoproteins differed markedly. ApoLP-Ala reassociated with all lipoproteins. ApoLP-Gin, reassociated in appreciable amounts only with high density lipoprotein, (though the amount of lipoprotein-lipid present in the incubation mixture of apoLP-Gln, with very low density or low density lipoprotein exceeded that present in the pure lipid mixture). Since the nature of the interaction of apoproteins with lipids of lipoproteins is not as yet understood, this observation remains unexplained. However, one may Biochim. Biophys. Acta, z80 (1972) g+Io.+ 104 S. zyxwvutsrqponmlkjihgfe EISENBERG et cd. speculate that the reassociation of apoproteins with lipoproteins may involve a specific interaction of proteiri with an organized lipoprotein macromolecule. Such a specificity may not be involved in the interaction of these same proteins with lipid emulsions. Additional evidence that part of the interaction of apoproteins with lipoproteins may involve a highly specific mechanism is found when the affinity of apoLPAla towards low density and high density lipoprotein is compared. The transfer of apoLP-Ala to high density lipoprotein far exceeded that to low density lipoprotein, though the low density lipoprotein mass (mol. wt zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR 2.2. zyxwvutsrqponmlkjihgfedcbaZYXWVUT IO” and zyxwvutsrqponmlkjihgfedcb 20~~ proteinZ3) in all experiments was 10-20 times greater than the high density lipoprotein mass (assumed mol. wt 2 ~106-4-10~ and 50% proteinzl). This difference in affinity may help explain the presence of only trace amounts of apoLP-Ala in low density lipoprotein9 as compared to very low density or high density lipoprotein2-5. We have already shown that the metabolism of apoLP-Glu and apoLP-Ala in very low density lipoprotein is different from that of apoLDLla. 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