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R O Ryan

Publications and source records attributed to R O Ryan.

At least 55 records · Page 3Linked to original sources
PubMed1995

Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.

To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Structural studies of Manduca sexta lipid transfer particle with apolipoprotein-specific antibodies.

Studies have been conducted to characterize structural and functional properties of Manduca sexta lipid transfer particle (LTP). LTP is a high molecular weight complex of three apolipoproteins and lipid that facilitates the transfer of lipids between lipoproteins and tissues and among lipoproteins in insect hemolymph. Rabbit polyclonal antibodies were raised against each of the three LTP apolipoproteins isolated by preparative electrophoresis. Immunoblot experiments demonstrated that they are apolipoprotein-specific and that LTP apolipoproteins are immunologically distinct polypeptides. Antibody-capture enzyme-linked immunosorbent assay characterization of apolipoprotein-specific IgG demonstrated that each of the three antibodies recognizes native LTP and provided information on the apparent affinity of the antibodies for LTP. Apolipoprotein-specific IgG were then compared in lipid transfer assays to examine the effect of antibody binding on LTP-mediated lipid transfer. Although each of the antibodies inhibited transfer activity, anti-apoLTP-II was capable of nearly abolishing activity at low IgG concentrations (< 26.7 micrograms IgG/micrograms LTP). In contrast, anti-apoLTP-I and anti-apoLTP-III IgG inhibited LTP activity only at much higher concentrations (> 133.3 micrograms IgG/micrograms LTP). These results indicate that apoLTP-II is a catalytically important apolipoprotein. In immunoprecipitation experiments, using 125I-labeled LTP, anti-holoLTP, anti-apoLTP-I, and anti-apoLTP-II were each able to immunoprecipitate all three LTP apolipoproteins while anti-apoLTP-III was not.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Structural and binding characteristics of the carboxyl terminal fragment of apolipophorin III from Manduca sexta.

The molecular basis of the interaction of apolipophorin III (apoLp-III), an exchangeable apolipoprotein from hemolymph of the sphinx moth. Manduca sexta, with lipoprotein surfaces and phospholipids was studied by investigating the structural and binding properties of the C-terminal fragment of the native protein. A 4K peptide, corresponding to the terminal helical segment of the native protein, was generated by cyanogen bromide treatment, purified by gel filtration and reverse-phase HPLC, and characterized by N-terminal sequencing and amino acid and mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the peptide in buffer indicated a predominantly unstructured state while addition of trifluoroethanol (TFE), a helix-inducing agent, resulted in an alpha-helical structure. Sedimentation equilibrium studies revealed that the 4K peptide was monomeric in buffer. The 4K peptide assumed an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS) and lysolecithin, but was unstructured in the presence of dimyristoylphosphatidylcholine, either when added to preformed vesicles or upon cosonication, indicating an ability to bind to detergent micelles but not to phospholipid bilayers. Unlike native apoLp-III, the 4K peptide did not confer protection against turbidity development to human low density lipoprotein upon incubation with phospholipase C, indicating an inability to interact with the surface of lipoproteins. Upon interaction with SDS micelles, both the 4K peptide and apoLp-III were resistant to urea-induced denaturation when compared to free apoLp-III, as evaluated by CD spectroscopy. The structural stability conferred upon interaction with detergents was similar for both the peptide and the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Factors affecting the stability and conformation of Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from the migratory locust, Locusta migratoria, represents the only full-length apolipoprotein whose three-dimensional structure has been solved. In the present study, spectroscopic methods have been employed to investigate the effects of deglycosylation (via endoglycosidase F treatment) and complexation with lipid on the stability and conformation of this protein. Addition of isolated lipid-free apoLp-III to sonicated vesicles of dimyristoylphosphatidylcholine (DMPC) resulted in the formation of relatively uniform disklike complexes with an average Strokes diameter of 13.5 nm. Flotation equilibrium experiments conducted in the analytical ultracentrifuge revealed a particle molecular mass of 588 500 Da. Chemical cross-linking and compositional analysis of apoLp-III.DMPC complexes indicated five apoLp-III molecules per disk and an overall DMPC:apoLp-III molar ratio of 122:1. Circular dichroism (CD) spectra of apoLp-III samples suggested a loss of alpha-helical structure upon deglycosylation, while complexation with DMPC did not significantly alter the helix content (estimated to be > 75%). Fluorescence spectroscopy revealed that the apoLp-III tryptophan fluorescence emission maximum was blue-shifted from 347 to 332 and 321 nm upon deglycosylation and complexation with DMPC, respectively. In quenching experiments with native apoLp-III, tryptophan residues were shielded from the positively charged quencher, CsCl. Increased exposure to KI, CsCl, and acrylamide was observed upon deglycosylation, whereas complexation with DMPC yielded lower Ksv values for KI and acrylamide and an increased value for CsCl versus native lipid-free apoLp-III. In guanidine hydrochloride denaturation studies monitored by CD or fluorescence, native, lipid-free apoLp-III displayed a denaturation midpoint of 0.60 M, and delta GDH2O = 5.37 kcal/mol was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗
PubMed1994

Binding of insect apolipophorin III to dimyristoylphosphatidylcholine vesicles. Evidence for a conformational change.

Apolipophorin-III (apoLp-III), a hemolymph protein of Manduca sexta, can reversibly associate with the surface of lipoprotein particles. In order to examine the lipid-associated form of apoLp-III, the present studies investigate the structure and properties of apoLp-III complexes with dimyristoylphosphatidylcholine (DMPC). Association of apoLp-III with DMPC vesicles results in the formation of uniform discs with an average diameter and width of 18.5 +/- 2.0 nm and 4.8 +/- 0.8 nm, respectively, as determined by electron microscopy. ApoLp-III.DMPC complexes analyzed by pore-limiting native gradient PAGE demonstrated that a single major species of complex was formed within a wide range of lipid to protein molar ratios (DMPC:apoLp-III; 13:1 to 360:1). Flotation equilibrium experiments, conducted in an analytical ultracentrifuge, confirmed that only one species of apoLp-III.DMPC complex was formed at an initial lipid to protein molar ratio of 67:1, with an apparent molecular mass of 642,000. Complexes cross-linked with dimethyl suberimidate indicate that there are a maximum of 6 apoLp-III molecules per disc. Circular dichroism experiments revealed that apoLp-III becomes essentially completely alpha-helical on formation of apoLp-III.DMPC complexes. Compared to apoLp-III in the lipid-free state, apoLp-III.DMPC complexes were relatively resistant to denaturation by guanidine HCl, displaying denaturation transitions with midpoints at 2.2 and 3.7 M guanidine HCl, respectively. The fluorescence excitation and emission spectra of apoLp-III.DMPC complexes demonstrate a large enhancement of tyrosine fluorescence as compared to the lipid-free state, suggesting that a conformational change occurs when apoLp-III associates with a lipid surface. Denaturation of apoLp-III in the complex by guanidine HCl resulted in a tyrosine fluorescence level similar to that of lipid-free apoLp-III in the presence of guanidine HCl. The tyrosine-induced fluorescence of the complex was quenched with both Cs+ (Kq = 0.573 M-1) and KI (Kq = 0.376 M-1). The results presented in this study indicate that the conformation of apoLp-III is stabilized when complexed with phospholipids and suggest that tyrosine fluorescence provides a sensitive method to detect M. sexta apoLp-III interaction with lipid surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Effect of phospholipase C and apolipophorin III on the structure and stability of lipophorin subspecies.

Four distinct subspecies of the insect hemolymph lipoprotein, lipophorin, that range in diacylglycerol (DAG) content from approximately 100 to 1000 molecules per particle, were treated with phospholipase C. Lipid analysis demonstrated that both phosphatidylcholine and phosphatidylethanolamine were hydrolyzed to DAG. Phospholipase C was used to remove 74-82% of the phospholipid of different lipophorins and these were analyzed for aggregation. Low density lipophorin (LDLp), the largest subspecies, with a diameter of approximately 23 nm, developed turbidity (monitored by sample absorbance at 340 nm) suggesting the formation of lipoprotein aggregates. High density lipophorin-adult (HDLp-A) and high density lipophorin-wanderer 1 (HDLp-W1) also displayed an increase in A340 when incubated with phospholipase C, although the maximal increase observed was considerably less than that for LDLp on a per particle basis. Phospholipase C caused only a minimal increase in A340 in a fourth subspecies, high density lipophorin-wanderer 2 (HDLp-W2), which contains an even lower amount of DAG. Electron microscopy was used to evaluate changes in particle morphology as a result of phospholipid depletion. HDLp-W2 and HDLp-W1 showed signs of progressive aggregation and particle fusion. A similar aggregation/fusion was seen in the case of high density lipophorin adult (HDLp-A) while LDLp samples contained multiple aggregation/fusion foci and resultant very large particles. In the presence of exogenous apolipophorin III (apoLp-III), phospholipase C-induced lipophorin aggregation/fusion was prevented. Electron microscopy of LDLp and HDLp-A samples revealed that apoLp-III-stabilized, phospholipase C-treated particles had a morphology similar to that of control particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Physical properties of apolipoprotein A-I from the chicken, Gallus domesticus.

The amphipathic alpha-helices of exchangeable apolipoproteins (apo) function to simultaneously facilitate interaction with lipid surfaces and the aqueous environment. In contrast to mammalian apoA-I's, which self-associate in the absence of lipid, chicken apoA-I, which shares 66% sequence homology with human apoA-I, exists as a monomeric protein when dissociated from high-density lipoprotein (HDL). Sedimentation equilibrium studies conducted in the analytical ultracentrifuge yielded a weight-average molecular weight of 28,170. Corresponding sedimentation velocity and diffusion experiments gave rise to s0(20,w) = 2.23 S and D0(20,w) = 6.39 x 10(-7) cm2/s. A translational frictional ratio (f/fmin) of 1.18 and an axial ratio of 4.0 were also determined from this data. The Stokes radius (Rs,sed = 2.80 nm) and translational frictional ratio were subsequently used to calculate estimated molecular dimensions of 25.2 x 100.8 A for chicken apoA-I. Circular dichroism (CD) studies revealed a highly alpha-helical structure predicted to be 74% by Provencher-Glöckner analysis. Denaturation studies performed on lipid-free apoA-I and monitored by CD revealed a midpoint of denaturation of 0.64 M guanidine hydrochloride. From plots of delta G(app) versus guanidine hydrochloride concentration, a delta GDH2O of 1.86 kcal/mol was determined. In other studies, a midpoint of temperature-induced denaturation for apoA-I of 57 degrees C was obtained. The effect of solvent pH on the secondary structure content of apoA-I revealed a significant loss of alpha-helix below pH 4.0 and above pH 10, suggesting that lipid-free apoA-I may by partially stabilized by the formation of intra- or interhelix salt bridges between oppositely charged amino acid side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Calorimetric and spectroscopic studies of the interaction of Manduca sexta apolipophorin III with zwitterionic, anionic, and nonionic lipids.

The nature of the interaction of apolipophorin III (apoLp-III) from the insect Manduca sexta with a variety of zwitterionic and anionic phospholipids and with several nonionic glycolipids was investigated by differential scanning calorimetry (DSC) and 31P-NMR spectroscopy. Monoglucosyldiacylglycerol, phosphatidylethanolamine, and phosphatidic acid (pH 7.2) appear to exhibit the weakest interaction with apoLp-III. DSC studies revealed that the gel/liquid-crystalline phase transition of these lipids is little affected by the binding of apoLp-III. Diglucosyldiacylglycerol, phosphatidylcholine (PC), phosphatidylserine, and phosphatidic acid (pH congruent to 8.8) seem to exhibit somewhat stronger interactions with apoLp-III. The binding of apoLp-III to these lipids induces the formation of lipid domains which melt less cooperatively and at higher temperatures than do the pure lipid dispersions, while having little effect on the melting enthalpy of lipid hydrocarbon chains. Phosphatidylglycerol (PG) and phosphatidic acid (pH > 9.3) appear to exhibit the strongest interactions with apoLp-III. The binding of apoLp-III to these lipids severely disrupts their bilayer structure, resulting in marked reductions in the cooperativity and enthalpy of the gel/liquid-crystalline phase transition of the lipids. Studies of binary mixtures of PC and PG indicate that such bilayer-disrupting interactions only occur in the presence of nonphysiologically high concentrations of PG. The binding of apoLp-III to binary mixtures of diacylglycerol and zwitterionic phospholipid has little effect on the chain-melting or the bilayer/nonbilayer phase transitions of these lipids, but it appears to promote the retention of water at the surface of the lipid aggregate. Our results indicate that the binding of apoLp-III to lipid bilayers is mediated primarily through polar and/or ionic interactions at the lipid bilayer surface. Our results also suggest that the interaction of apoLp-III with lipid bilayers promotes the hydration of their surfaces, a property which is consistent with the proposed in vivo functions of this protein.

Animals↗
PubMed1993

Conformational, thermodynamic, and stability properties of Manduca sexta apolipophorin III.

Apolipophorin III (apoLp-III) is a major protein in hemolymph of adult Manduca sexta. Although it normally exists in a lipid-free state, during sustained flight, apoLp-III functions as an apolipoprotein, reversibly associating with the surface of lipoprotein particles. In an effort to gain a fuller understanding of this dual existence, we have investigated its solution properties using spectroscopic methods. The UV absorption spectrum of apoLp-III is distinctive owing to the absence of tryptophan and the presence of a single tyrosine residue. Circular dichroism experiments revealed an enhancement of apoLp-III alpha-helical content when spectra were obtained in 50% trifluoroethanol versus aqueous buffer. The helical content in buffer was unaffected by protein concentration, suggesting that apoLp-III exists in solution as a monomeric species. At pH values > 10 and < 4, there was a marked loss of helical content. Increasing the temperature of apoLp-III solutions also caused a loss of secondary structure, with a temperature-induced denaturation midpoint of 52 degrees C. Upon recooling of heat-denatured apoLp-III, approximately 95% of the secondary structure was restored. In guanidine HCl denaturation studies monitored by CD, a 50% transition midpoint of 0.355 M was determined, corresponding to a delta GDH2O of 1.29 kcal/mol. Fluorescence studies indicated that guanidine HCl induced an enhancement of tyrosine fluorescence emission at 300 nm when excited at 277 nm. In native apoLp-III, we propose that tyrosine fluorescence is quenched to a large extent due to a hydrophobic stacking interaction of its side chain with that of a neighboring phenylalanine residue. delta GDH2O was determined from the fluorescence data to be 2.1 kcal/mol, with a transition midpoint occurring at 0.25 M guanidine HCl. The lower concentration of guanidine HCl required to induce half-maximal tyrosine fluorescence enhancement versus the transition midpoint detected by CD may be a reflection of the fact that this residue is located near the COOH-terminal end of the protein and as such may be more susceptible to denaturation. The results presented indicate that apoLp-III assumes a relatively labile conformation in solution that appears to be partially stabilized by side chain charge-charge interactions within predicted alpha-helical segments.

Animals↗
PubMed1993

Prevention of phospholipase-C induced aggregation of low density lipoprotein by amphipathic apolipoproteins.

Phospholipase C (PL-C) digestion of human low density lipoprotein (LDL) results in hydrolytic cleavage of the phosphocholine head group of phosphatidylcholine, thereby generating diacylglycerol. Loss of amphiphillic surface lipids and/or accumulation of diacylglycerol causes LDL samples to develop turbidity. Examination of PL-C treated LDL by electron microscopy revealed a progressive aggregation of LDL as a function of phosphatidylcholine hydrolysis: fused particles, clusters, and multiple stacked aggregates were observed. Lipid analysis of untreated and aggregated LDL confirmed that the phosphatidylcholine content of the latter had decreased with a corresponding increase in diacylglycerol. It is likely that phospholipolysis created hydrophobic gaps within the surface monolayer of LDL, thereby inducing LDL fusion and aggregation. When amphipathic alpha-helix-containing apolipoproteins, such as human apoA-I or Manduca sexta apolipophorin III (apoLp-III) were present, PL-C treated LDL did not aggregate. Compositional analysis of apolipoprotein-containing PL-C LDL showed that phospholipolysis was not affected by the presence of apolipoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipoproteins re-isolated following incubation with PL-C revealed an association of apoA-I or apoLp-III with PL-C digested LDL. Electron microscopy showed no major morphological differences between native LDL and apoprotein stabilized PL-C treated LDL and the average particle diameter of apoA-I stabilized PL-C LDL was 22.5 +/- 2.2 nm versus 22.8 +/- 1.6 nm for control LDL. Incubation of tritium-labeled apoLp-III with LDL and PL-C demonstrated that association of apoLp-III with PL-C LDL correlated with the extent of phospholipid hydrolysis, the apolipoproteins apparently being recruited to compensate for the increased hydrophobic surface created by conversion of phosphatidylcholine into diacylglycerol. The results suggest that transient association of amphipathic apolipoproteins with damaged or unstable LDL may provide a mechanism to obviate formation of atherogenic LDL aggregates in vivo.

Animals↗
PubMed1992

31P-NMR study of the phospholipid moiety of lipophorin subspecies.

31P-NMR spectra of four distinct subspecies of Manduca sexta hemolymph lipophorin revealed the presence of two resonances separated by 0.6 ppm. Phospholipid analysis of the lipoproteins showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present and their mass ratio correlated well to the intensity of the two resonances in each of the different subspecies. The two resonances persisted in 31P-NMR spectra of organic solvent extracts of lipophorin. These results, together with the fact that PE, but not PC, can form an intramolecular hydrogen bond between the phosphate oxygen and the amino group of ethanolamine, resulting in deshielding of the phosphorus nucleus (and a 0.6 ppm downfield shift), strongly suggest the resonances observed represent the PC and PE components of these lipoproteins. 31P-NMR line-width data obtained as a function of temperature and solvent viscosity were used to calculate the chemical shift anisotropy (delta sigma), intrinsic viscosity (eta'), and lateral diffusion coefficients (DT) of PC and PE in different lipophorin subspecies. eta' and DT for PC and PE were similar among high-density lipophorins but differed in low-density lipophorin (LDLp). These differences may be related to the large increase in diacylglycerol content in this particle and/or the association of up to 16 molecules of apolipophorin III. On the basis of the known lipid compositional differences between LDLp and high-density lipophorin subspecies, we propose that uptake of large amounts of diacylglycerol during LDLp formation results in partitioning of this lipid to the surface monolayer where it intercalates between phospholipid molecules. Diacylglycerol intercalation creates gaps between phospholipid head groups that expose the hydrophobic surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Insect lipid transfer particle can facilitate net vectorial lipid transfer via a carrier-mediated mechanism.

The mechanism of facilitated lipid transfer by insect or mammalian plasma lipid transfer proteins has not been elucidated. Transfer catalysts may act as carriers of lipid between donor and acceptor lipoproteins or, alternatively, transfer may require formation of a ternary complex. This study was designed to determine if Manduca sexta hemolymph lipid transfer particle (LTP) can facilitate net vectorial transfer of lipid without concomitant contact between donor and acceptor lipoproteins and LTP. M. sexta [3H]diacylglycerol-high density lipophorin-larval ([3H]DAG-HDLp-L) and human low density lipoprotein (LDL) were covalently bound to Sepharose matrices and packed into separate columns. In incubations lacking LTP, greater than 98% of the recovered DAG remained associated with HDLp-L. An unrelated hemolymph storage protein, arylphorin, was unable to catalyze the transfer of DAG between solid-phase lipoproteins. Facilitated transfer of DAG from HDLp-L to LDL was observed when LTP was circulated between the columns. Under these conditions, facilitated transfer occurred at a rate of 2.24 ng of DAG/h (versus 0.16 microgram of DAG/h in the control), and after 16 h greater than 26% of recovered labeled DAG was transferred to LDL. This corresponds to a 14-fold rate enhancement induced by LTP. The LTP-specific transfer of DAG between physically separated lipoproteins demonstrates the ability of LTP to facilitate net lipid transfer via a carrier-mediated mechanism in the absence of a ternary complex involving donor, acceptor, and catalyst. In experiments aimed at assessing the relative contribution of ternary complex formation to DAG transfer, acceptor LDL was circulated with HDLp-L remaining immobilized. Under these conditions, LTP induced a 13-fold rate enhancement from 1.3 to 16.3 micrograms of DAG/h. The similar rate enhancements observed with both lipoproteins bound and only donor bound suggest the overall contribution of ternary complex formation to facilitated lipid transfer is insignificant. The described system should prove useful in mechanistic studies of other transfer proteins as well as studies of transfer of other lipids.

Animals↗
PubMed1992

Human apolipoprotein A-I liberated from high-density lipoprotein without denaturation.

Apolipoprotein A-I (apoA-I) was liberated from human high-density lipoprotein (HDL) without exposure to organic solvents or chaotropic salts by the action of isolated insect hemolymph lipid transfer particle (LTP). LTP-catalyzed lipid redistribution results in transformation of HDL into larger, less dense particles accompanied by an overall decrease in HDL particle surface area:core volume ratio, giving rise to an excess of amphiphilic surface components. Preferential dissociation of apolipoprotein versus phospholipid and unesterified cholesterol from the particle surface results in apolipoprotein recovery in the bottom fraction following ultracentrifugation at a density = 1.23 g/mL. ApoA-I was then isolated from other contaminating HDL apolipoproteins by incubation with additional HDL in the absence of LTP, whereupon apolipoprotein A-II and the C apolipoproteins reassociate with the HDL surface by displacement of apoA-I. After a second density gradient ultracentrifugation, electrophoretically pure apoA-I was obtained. Sedimentation equilibrium experiments revealed that apoA-I isolated via this method exhibits a tendency to self-associate in an aqueous solution while its circular dichroism spectrum was indicative of a significant amount of alpha-helix. Both measurements are consistent with that observed on material prepared by denaturation/renaturation. The ability of apoA-I to activate lecithin:cholesterol acyltransferase was found to be similar to that of apoA-I isolated by conventional methods. The present results illustrate that LTP-mediated alteration in lipoprotein particle surface area leads to dissociation of substantial amounts of surface active apoprotein components, thus providing the opportunity to isolate apoA-I without the denaturation/renaturation steps common to all previous isolation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.

Animals↗
PubMed1992

Primary structure of locust flight muscle fatty acid binding protein.

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16-23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT-NotI primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.

Amino Acid Sequence↗
PubMed1992

A turbidimetric assay of lipid transfer activity.

A novel method to assay insect plasma lipid transfer particle (LTP) activity has been developed that employs insect high density lipophorin (HDLp) and human low density lipoprotein (LDL) as donor/acceptor substrate particles. At a 3:1 or greater HDLp:LDL protein ratio, LTP-mediated net vectorial transfer of diacylglycerol from lipophorin to LDL produces destabilized LDL particles that aggregate, causing sample turbidity. Turbidity was measured spectrophotometrically as a function of absorbance at 340 nm. After an initial lag phase, lipoprotein sample turbidity increased as a function of reaction time and LTP concentration. Saturation was observed at longer times or higher LTP concentrations, indicating that a reaction end point had been reached. As the substrate HDLp concentration was increased relative to LDL, a saturable increase in LTP-induced lipoprotein sample turbidity was observed. When the LDL concentration was increased relative to HDLp, however, there was an initial production of turbidity but at higher concentrations the sample did not develop turbidity. Reaction progress was also dependent on temperature over the range 0-37 degrees C. Taken together the results are consistent with the concept that LTP-mediated diacylglycerol transfer from HDLp to LDL creates unstable product LDL particles that aggregate. The assay method is advantageous because it employs relatively abundant, natural lipoprotein substrates, does not require prelabeling of donor lipid particles with radioactive or fluorescent lipids, and does not require separation of donor and acceptor after incubation. This is the first description of a lipid transfer assay that can be measured spectrophotometrically.

Animals↗
PubMed1992

Effect of particle lipid content on the structure of insect lipophorins.

Four distinct subspecies of the major insect lipoprotein, lipophorin, that range in overall lipid content from 20 to 51% of the particle mass, were isolated from the hemolymph or oocytes of the tobacco hornworm, Manduca sexta. Examination of these subspecies by electron microscopy revealed distinctive morphologies. Adult high density lipophorin (HDLp-A) was found to be an approximately spherical particle with a diameter of 15 +/- 1 nm, while HDLp-Wanderer 1 (W1), was more rectangular in shape and had a distinct cleft extending into the particle at one end. In the case of HDLp-Wanderer 2 (W2) the cleft was deeper and wider than that in HDLp-W1. In egg very high density lipophorin (VHDLp-E) the cleft was increased in size to the extent that the particle had an overall crescent-like conformation. Circular dichroism spectroscopy of the three lipophorin subspecies that contain only apolipophorin I and II revealed that only minor differences in the global protein secondary structure occur as the particle lipid content is decreased. The VHDLp-E apolipoproteins are an exception in that, while having the same alpha-helix content as HDLp-W1 and HDLp-W2, they contain less beta-structure and correspondingly more random coil. Limited digestion of the apolipoprotein components of the lipophorin subspecies with trypsin revealed that as the lipid content of the particles decreases the susceptibility of the apolipoprotein to proteolytic degradation increases. Likewise, tryptophan fluorescence quenching experiments demonstrated that the relative exposure of lipophorin apolipoprotein tryptophan residues also increases as the particle lipid content decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗