Abstract
Chemical reduction of human plasma lipoprotein(a) (Lp(a)) yielded two water-soluble products which were separated by rate zonal ultracentrifugation. Apolipoprotein(a) (apo(a)) was completely recovered from the bottom of the gradient, whereas lipoprotein(a-) (Lp(a-)), which contained all of the lipids and apo-B100 of Lp(a), floated. By the techniques of circular dichroism and viscometry Lp(a-) was identical to low density lipoprotein (LDL). Lp(a-) was slightly larger in mass than autologous LDL and contained proportionally more triglyceride. The difference in mass between Lp(a) and Lp(a-) was accounted for by the loss of 2 molecules of apo(a) from the Lp(a) particle. The molecular weight of reduced and carboxymethylated apo(a) was 281,000 as determined by sedimentation equilibrium in 6 M guanidine HCl. By circular dichroism the structure of apo(a) was mostly random (71%) with the remainder representing 8% alpha-helix and 21% beta-sheet; its intrinsic viscosity, 28.3 cm3/g, was consistent with an extended flexible coil. The amino acid composition was characterized by an unusually high content of proline (11.4 mol %) as well as tryptophan, tyrosine, arginine, threonine, and a low amount of lysine, phenylalanine, and isoleucine. Apo(a) contained 28.1% carbohydrate by weight represented by mannose, galactose, galactosamine, glucosamine, and sialic acid in an approximate molar ratio of 3:7:5:4:7, respectively. Overall, the structure of Lp(a) appears to be consistent with a rigid spherical LDL-like core particle which, as a consequence of its association with a flexible glycoprotein such as apo(a), favors the entrapment of significant amounts of hydrodynamically associated solvent. Furthermore, the Lp(a-) remnant generated by the removal of apo(a) from Lp(a) was similar in structure but not identical to autologous LDL.
Highlights
From the Departments of $Medicine, TBiochemistry,and Molecular Biology, The Pritzker Schoolof Medicine, The University of Chicago, Chicago,Illinois 60637
In thecontext of bottom of thgeradientw, hereas lipoprotein(a-) the current studies we considered it of interest to establish (Lp(a-)), which containedall of the lipids and apo-Bloothe physicochemical relationships between Lp(a-) and autimLolsihfppmoaLo(rnaappe)tnr(raoaadiut)gnev,tlodiiynflscolLco(egopLarom(tiDuaede-sdeL)t..r)TBLy.whyDLLeatLppdsh((ieaafaaf-c-t)ne)ecrdcowewhunacanncsositeqensiudntliaidemgfisenohanoertstlfdsiypbbccyreailoratlcwptrhugoteleoereatrnirloolionsndswaicmlolhyfadrso2es-nsitdtbctohoeeylicutosealgrdauompsaueoirsnilosieLpfedoDtfhpirLtoersomaptienbrtioshneopenrepdccrleatoeiryueospsftloipiinnnhagtyshoossefriecdsaosseptcrtrothouhe(ceamut)unpicrdoteaoseoLlsrfdiDsbaLtLiatlpani.t(Fdyoaun)ttr.hhtahaepreotorlL(meapto)(hwraae)et, molecules of apo(a) from the Lp(a) particle. mThoelecular weight of reduced and carboxymethylated apo(a) was 281,000as determined by sedimentationequilib- Isolation of Plasma Lipoproteins-Two healthy female subjects rium in 6 M guanidine HCl
The abbreviations are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a), which is cross-linked to apo-B of Lp(a) through disulfide bonds; Lp(a-), remnant lipoprotein particle obtained upon the reduction and subsequent removal of apo(a) from Lp(a); LDL, low density lipoprotein; Na2EDTA,sodium salt of ethylenediaminetetraacetic acid CD, circular dichroism; GuHCl, guanidine HCl; NeuNac, monitor
Summary
0 1986by The American Societyof Biological Chemista, Inc. Vol 261, No 19. Galactosamine, glucosamine, and sialic aciidn an were obtained by adjusting plasma to d 1.21 g/ml with solid NaBr waOpivptehrroaaxllri,imgthiadetemssptohrluearcrirtcuaatrlieoofLLDpLo(f-al)i3k:ae7p:pc5eo:4ar:ers7,ptaroertsibpceelectciowvnehsliayicss.hte, naaa1tsn5cdpo"rmCce.evbLniiontDruaiLtsfiuloyagnndindoegfscLrtrahpitbe(emeazd)wo(inen2ra)el.aaisn6o0dladTteeindsrfoirttoyomrgrtaahtdei5e9tno,t0tu0a0lltlrirappcomepnrtofrotiefruin2gs0autihsoinnasgt a consequence of its association with a flexible glyco- Isolation of Apo(a) from Lp(a)-Apo(a) was isolated from Lp(a) as protein such as apo(a), favors the entrapment of sig- previously described [1].In brief, solutions of Lp(a) in 0.15 M NaCl, nificant amounts of hydrodynamically associated sol- 0.01% Na2EDTA and NaN3, pH 7.0, typically containing approxivent. The abbreviations are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a), which is cross-linked to apo-B of Lp(a) through disulfide bonds; Lp(a-), remnant lipoprotein particle obtained upon the reduction and subsequent removal of apo(a) from Lp(a); LDL, low density lipoprotein; Na2EDTA,sodium salt of ethylenediaminetetraacetic acid CD, circular dichroism; GuHCl, guanidine HCl; NeuNac, monitor. ChemicalAnalysis-Protein content was determined by the method of Lowry et ai. [16] using bovine serum albumin as standard as previously described [7].Phospholipid [17],cholesterol, both free and esterified [18, 19], and triglyceride [20] analyses were performed as described previously [21]
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