Chain length-function correlation of amphiphilic peptides. Synthesis and surface properties of a tetratetracontapeptide segment of apolipoprotein A-I

The segment corresponding to residues 121 to 164 of human plasma apolipoprotein A-I (apo-A-I) has been synthesized by the Merrifield solid phase method. The peptide binds to unilamellar phospholipid vesicles and to phospholipid-cholesterol mixed vesicles. The surface affinity of the peptide measured in this way indicated that the mechanism of binding is the same as that of apo A-I (144-165) and apo A-I itself. The peptide appears to be a globular monomer in a aqueous solution, with 17% alpha helix content. The peptide bound to vesicles activates lecithin:cholesterol acyltransferase: compared to apo A-I, the peptide is about 30% as efficient in the activation of cholesterol esterification and of phospholipid hydrolysis when the surface is saturated by the activator. For a variety of amphiphilic peptides and for apo A-I, the lecithin: cholesterol acyltransferase-activating ability correlates well with their alpha helix contents in 50% trifluoroethanol.     

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.     

Rabbit apolipoprotein A-I mRNA and gene. Evidence that rabbit apolipoprotein A-I is synthesized in the intestine but not in the liver

In order to study the tissue-specific expression of rabbit apolipoprotein (apo) A-I, a 923-base-pair clone, pRBA-502, complementary to rabbit apo A-I mRNA was identified from a rabbit intestinal cDNA library by hybrid-select translation and immunoprecipitation methods. Northern blot and dot-blot hybridization, utilizing 32P-labeled pRBA-502, revealed that the rabbit apo A-I gene is expressed in the intestine, not in the liver and that rabbit apo A-I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA-502 has been determined and the complete amino acid sequence of the corresponding apo A-I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1-18 and 19-24 of the primary translation product represent the presegment and prosegment, respectively, of apo A-I. Matured rabbit apo A-I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA-502 as a probe, a 15.5-kb genomic fragment, which contains the entire apo A-I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5' upstream flanking region of the rabbit and human apo A-I genes showed 78% sequence homology. Like the human apo A-I gene, the rabbit apo A-I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A-I gene is identical to that of pRBA-502. Our data showed that the lack of expression of apo A-I gene in rabbit liver is not due to the alternation of rabbit liver apo A-I gene sequence and suggest that the expression of apo A-I gene in rabbit liver is regulated by a trans-acting regulating element(s).     

Apolipoprotein A-IGiessen (Pro143----Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase

Apolipoprotein A-IGiessen is a variant form of apo A-I that is displaced from the corresponding normal A-I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501-507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A-I in plasma represented by the A-IGiessen in these subjects ranged over 25-30%. The variant and normal major A-I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A-I was replaced by an arginine in the variant A-I at residue 143. The ability of the mutant A-I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A-I was about 60-70% of that demonstrated by control A-I. Residue 143 is in a putative beta-turn between two of the repeating amphiphilic helices in apolipoprotein A-I and may be a critical determinant of the protein's structure and function.     

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.     

Reduction of lipid hydroperoxides by apolipoprotein B-100.

We have previously isolated two proteins which can reduce phosphatidylcholine hydroperoxide (PC-OOH) from human blood plasma and identified one of the proteins as apolipoprotein A-I (Mashima, R. , et al. (1998) J. Lipid Res. 39, 1133-1140). In the present study we have identified the other protein as apolipoprotein B-100 (apo B-100) by amino acid sequence analysis of its tryptic peptides. The reactivity of lipid hydroperoxides with apo B-100 decreased in the order of PC-OOH > linoleic acid hydroperoxide > cholesteryl ester hydroperoxide under our experimental conditions. Pretreatment of apo B-100 with chloramine T, an oxidant of methionine, diminished the PC-OOH-reducing activity, indicating that some of 78 methionines are responsible for the reduction of PC-OOH. Despite the presence of 6 methionines in albumin, albumin was inactive to reduce PC-OOH. Free methionine was also inactive. These data suggest that the accessibility and binding of lipid hydroperoxides to the protein methionine residues are crucial for reduction of lipid hydroperoxides.     

Acid Lipase Inhibitor in Chicken Plasma Identified as Apolipoprotein A-I

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895–898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken.

The purified inhibitor migrated with the same mobility on SDS–PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.     

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms

The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.     

The core protein of alphaviruses. 1. Purification of peptides and complete amino-acid sequence of Semliki Forest virus core protein

The primary structure of the core protein of Semliki Forest virus has been established by protein chemical characterization of 102 peptides, generated by digestion with trypsin, pepsin, thermolysin, and by partial acid cleavage of the protein. Besides a difference in one position, the sequence as established by these experiments is in agreement with the sequence predicted from the nucleotide sequence of the mRNA [Garoff et al. (1980) Proc. Natl Acad. Sci. USA, 77, 6376-6380]. The core protein has a blocked N terminus, consists of 267 amino acid residues, and has the following amino acid composition: Asp12, Asn9, Thr16, Ser10, Glu11, Gln15, Pro23, Gly20, Ala23, Val19, Met8, Ile11, Leu9, Tyr7, Phe6, His7, Lys37, Arg15, Trp5, Cys4, and an Mr of 29919. It contains 22.1% basic amino acids, mainly lysines, compared with a total of 8.6% acidic residues. The resulting surplus of positive charge is located in the N-terminal half of the protein (predominantly arginines at positions 12-21 and lysines at positions 66-114). Other amino acids are also unevenly distributed; proline and glutamine are accumulated in the N-terminal half of the sequence whereas histidine, glycine and the acidic residues are mainly present in the C-terminal part. This distribution suggests that the virus core protein consists of two or more structural domains.     

Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A(2) activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A(2) activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices alpha 3;-4 and alpha His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant.These data suggest that residues T123 and F382, located N-terminal of helices alpha 3-4 and alpha His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

The complete amino acid sequence of proapolipoprotein A-I of chicken high density lipoproteins

The complete amino acid sequence of proapolipoprotein (proapo) A-I of chicken high density lipoproteins was determined by sequencing overlapping peptides produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage. There are 240 amino acid residues in mature chicken apoA-I. By direct sequence analysis of a cyanogen bromide peptide, we also determined the sequence of a 6-amino-acid prosegment which is present at approx. 10% the molar amount of the mature peptide in chicken plasma. Sequence comparison among apoA-I from chicken, human, rabbit, dog and rat, and secondary structure analysis indicate that while the degree of sequence homology is only moderate (less than 50% between chicken and man), there is good conservation of apoA-I secondary structure, especially in the N-terminal two-thirds of the protein in these widely separated species.     

Molecular dynamics simulations on discoidal HDL particles suggest a mechanism for rotation in the apo A-I belt model

Apolipoprotein A-I (apo A-I) is the major protein component of high-density lipoprotein (HDL) particles. Elevated levels of HDL in the bloodstream have been shown to correlate strongly with a reduced risk factor for atherosclerosis. Molecular dynamics simulations have been carried out on three separate model discoidal high-density lipoprotein particles (HDL) containing two monomers of apo A-I and 160 molecules of palmitoyloleoylphosphatidylcholine (POPC), to a time-scale of 1ns. The starting structures were on the basis of previously published molecular belt models of HDL consisting of the lipid-binding C-terminal domain (residues 44-243) wrapped around the circumference of a discoidal HDL particle. Subtle changes between two of the starting structures resulted in significantly different behavior during the course of the simulation. The results provide support for the hypothesis of Segrest et al. that helical registration in the molecular belt model of apo A-I is modulated by intermolecular salt bridges. In addition, we propose an explanation for the presence of proline punctuation in the molecular belt model, and for the presence of two 11-mer helical repeats interrupting the otherwise regular pattern of 22-mer helical repeats in the lipid-binding domain of apo A-I.     

Primary structure of Beijing duck apolipoprotein A-1

The primary structure of Beijing duck apolipoprotein A-1 was determined by sequencing peptide fragments derived from tryptic and endoproteinase Asp-N digestion of the protein, and alignment with homologous chicken apo A-1. All of the peptide fragments were isolated by high-pressure liquid chromatography (HPLC) with a Vydac C18 column using a trifluoroacetic acid (TFA) buffer system. The N-terminus of the protein was determined to be aspartic acid by directly sequencing 52 residues of the intact protein. The C-terminus was alanine. The protein contains 240 amino acid residues. By analysis of the whole protein and its tryptic peptides, a six amino acid (Arg-Tyr-Phe-Trp-Gln-His) prosegment was determined. No cross-reactivity between duck and human apo A-1 with a goat antiserum against human apo A-1 was found. Sequence analysis of apo A-1 of other species indicates that amino acid substitutions in rat are more extensive than in other mammals. Isoleucine residues in apo A-1 are inversely correlated to the homology of human to other species, except dog.     

The partial sequence of two large peptides from the N-terminal half of heavy chains from normal rabbit immunoglobulin G

The partial amino acid sequence of two large peptides is described. These were prepared from the N-terminal half of the heavy chain of immunoglobulin G from pooled normal rabbit serum by tryptic digestion after the in-amino groups of the lysine residues had been blocked with S-ethyl trifluorothioacetate. These peptides are believed to account for about 145 residues of fragment C-1, the N-terminal section of rabbit immunoglobulin G heavy chain prepared by cyanogen bromide cleavage. The evidence from the present paper and the preceding paper (Cebra, Givol & Porter, 1968) suggests that it may be possible to deduce a predominant amino acid sequence for most, if not all, of this section of the molecule.     

Human spleen histone H1. Isolation and amino acid sequence of a main variant, H1b

The complete amino acid sequence of a main variant, H1b, of human spleen histone H1 was determined, following previous determinations of human spleen histones H2B, H2A, H3, and H4. High-performance liquid chromatography on C8 silica of the H1 fraction yielded the homogeneous H1b subfraction; this variant was estimated to account for 60% of the total of the four H1 variants. The sequence determination was performed with four main fragments, I to IV, obtained by limited chymotryptic digestion of H1b. Together with direct sequencing by automated Edman degradation of fragments II, III, and IV, fragment I, blocked at the N-terminal, and fragment IV, the C-terminal half the H1b molecule, were sequenced after further digestion with staphylococcal protease and others. The four fragments were aligned with three overlapping peptides each derived from chymotryptic partial fragments, I-II and I-II-III, and intact H1b. Carboxypeptidase digestion of intact H1b confirmed the C-terminal sequence of the molecule. Thus, the total sequence of H1b was completely determined; it consists of a total of 218 amino acid residues, has a molecular weight of 21,734 in the unmodified form, and is completely acetylated at the N-terminal serine residue and partially methylated at the lysine residue 25. This sequence is compared with two mammalian somatic H1 sequences.     

Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

The amino acid sequence of the ribosomal protein S8 of Escherichia coli.

The primary structure of protein S8 from the 30S subunit of Escherichia coli ribosomes has been determined by sequencing the peptides derived from tryptic, chymotryptic, thermolytic and staphylococcal protease digestion of the protein. Protein S8 has 129 amino acid residues which result in a molecular weight of 13996. The N-terminal part of the sequence up to position 68 is in complete agreement with the reported sequence data[1,2]. However, differences exist in the C-terminal half, where an additional hydrophobic tryptic peptide has been found.