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J I Gordon

Publications and source records attributed to J I Gordon.

At least 217 records · Page 12Linked to original sources
PubMed1988

Expression of human preproapo AI and pre(delta pro)apoAI in a murine pituitary cell line (AtT-20). A comparison of their intracellular compartmentalization and lipid affiliation.

The role of the NH2-terminal propeptide of human apolipoprotein (apo) AI in intracellular transport and lipid-protein interactions was examined by transfecting a murine anterior pituitary cell line (AtT-20) with the human preproapo AI gene and a mutant gene lacking the 18-base pair segment of exon III encoding its hexapeptide prosegment. ProapoAI was not processed to the mature apolipoprotein either prior to or after export from these cells making this an attractive model system for directly assessing structure/activity relationships of its propeptide. ApoAI was sorted into a regulated pathway for protein export. The signal responsible for this trafficking pattern was not contained in the prosegment since both pro- and mature ApoAI exhibited a similar rate of secretion, a similar magnitude of stimulation of export by the secretagogue 8-bromo-cyclic AMP, and similar targeting to dense core granules. This sorting behavior, exhibited by a protein which is not normally targeted to dense core secretory granules in its cells of origin, raises questions about the domains and mechanisms involved in protein sorting into the regulated and constitutive pathways of AtT-20 cells. Density gradient ultracentrifugation, immunoaffinity chromatographic and electron microscopic analysis indicated that approximately 10% of proapoAI and approximately 10% apoAI appeared in AtT-20 culture media in the form of two discrete classes of nascent lipoproteins: a small 6-8 nm spherical particle and a larger discoidal particle. These particles had morphologies identical with those secreted by Hep G2 cells which normally synthesize apolipoproteins. Although these results do not resolve the issue of whether or not a fraction of apoAI can act as an acceptor of cellular lipid during its transport through the secretory pathway, the data do show that this functional capability for lipoprotein assembly is not obviously regulated by its prosegment.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.

Amino Acid Sequence↗
PubMed1988

The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-A resolution.

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant cytoplasmic protein which is synthesized in the small intestinal lining cell where it is thought to participate in the absorption and intracellular metabolism of fatty acids. Each mole of this 132-residue polypeptide binds 1 mol of long chain fatty acid in a noncovalent fashion. Because of its small size and single ligand-binding site, I-FABP represents an attractive model for defining the molecular details of long chain fatty acid-protein interactions. The structure of Escherichia coli-derived rat I-FABP has now been solved to 2.5 A resolution using three isomorphous heavy atom derivatives. The protein consists of 10 anti-parallel beta-strands present as two orthogonal beta-sheets. Together a "clam shell-like" structure is formed with an opening located between two beta-strands and an interior that is lined with the side chains of nonpolar amino acids. The bound fatty acid ligand is located in the interior of the protein and has a bent conformation, possibly reflecting the presence of several gauche bonds in the hydrocarbon tail. Our present interpretation of the electron density map suggests that the fatty acid is oriented with its carboxylate group facing the guanidinium group of Arg127, whereas the end of its hydrocarbon tail is in close proximity to Val106. The indole side chain of Trp83 forms the molecular framework around which the principal bend of the hydrocarbon chain occurs.

Amino Acid Sequence↗
PubMed1988

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase.

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.

Acyltransferases↗
PubMed1988

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities.

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.

Acyl Coenzyme A↗
PubMed1988

Substrate specificity of eukaryotic signal peptidase. Site-saturation mutagenesis at position -1 regulates cleavage between multiple sites in human pre (delta pro) apolipoprotein A-II.

We have previously described a novel mutant of human preproapolipoprotein A-II (pre(delta pro)apoA-II) in which the wild-type 18-amino acid-long signal sequence (Gly18 decreases) was functionally redefined to 20 amino acids in length (Ala 20 decreases). We have used this mutant as a model preprotein to probe the substrate specificity of eukaryotic signal peptidase. Site-saturation mutagenesis was performed resulting in the substitution of 13 different amino acids (acidic, basic, aromatic, hydrophobic, and small-neutral) for Ala20 (or position -1). The effects of these substitutions were assessed using an in vitro transcription/translation/microsomal membrane processing system. NH2-terminal sequence analysis of the 13 mutant proteins demonstrated that amino acids which occupy position -1 in a signal peptide are critical in establishing a good context for signal peptidase cleavage and that two or more potential sites of cleavage may compete for recognition by signal peptidase.

Amino Acid Sequence↗
PubMed1988

The hypothyroid (hyt/hyt) mouse: a model system for studying the effects of thyroid hormone on developmental changes in gene expression.

Thyroid hormone has been implicated as an important factor in rodent development. We have used a strain of mice with a recessive mutation producing congenital primary hypothyroidism (C.RF/J-hyt/+) to study the effects of thyroid hormone on developmental changes in the expression of genes encoding a number of proteins involved in lipid metabolism and transport. Total cellular RNA was prepared from the small intestine and liver of hyt/hyt mice and their unaffected littermates (+/?) at various times during postnatal development. RNA blots were probed with apolipoprotein A-I, A-II, A-IV, B, and E cDNAs plus cDNAs encoding the low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and three cytoplasmic hydrophobic ligand-binding proteins (two fatty acid-binding proteins and a protein that binds all-trans-retinol). Hypothyroidism results in small changes (1.5- to 5-fold) in the concentration of many of these mRNAs in liver and small intestine between postnatal days 15 and 50. A much greater tissue-specific effect was noted on apolipoprotein B (apoB) gene expression. In euthyroid +/? animals, apoB mRNA levels fall by a factor of 30 in liver between days 20 and 35 without a comparable decrease in the small intestine. This liver-specific decrease does not occur in hyt/hyt animals. The normal decrease in hepatic apoB mRNA levels is accompanied by a decrease in plasma apoB-100 but not apoB-48. No reduction in either form of plasma apoB was noted in hyt/hyt animals. Mutant hyt/hyt mice given thyroxine from birth to 35 days had liver apoB mRNA levels comparable to those in +/? littermates. In contrast, hepatic apoB mRNA concentrations did not fall to normal levels in hyt/hyt mice given thyroxine from postnatal days 15 to 35. All treatment groups have comparable levels of plasma corticosteroids. These data suggest that (i) there is a critical period or a required response time during postnatal development for thyroid hormone action on apoB gene expression, (ii) thyroid hormone's effect on apoB is tissue specific, and (iii) the hyt/hyt mouse represents a useful system to evaluate the developmental effects of thyroid hormone on specific gene expression.

Animals↗
PubMed1988

Heteroatom-substituted fatty acid analogs as substrates for N-myristoyltransferase: an approach for studying both the enzymology and function of protein acylation.

Myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that transfers the myristoyl (14:0) moiety from myristoyl CoA thioester to nascent proteins, is remarkably specific for both peptide and fatty acyl CoA substrates. To investigate the interaction of NMT with fatty acyl CoA substrates, we have synthesized 10 oxygen- or sulfur-substituted fatty acid analogs. These analogs differ dramatically in hydrophobicity from naturally occurring fatty acids of similar length. As acylpeptides, sulfur-substituted myristic acid analogs migrate on reverse-phase HPLC like 11:0 or 12:0 fatty acids, while oxygen-substituted analogs migrate like 9:0 to 11:0 fatty acids. CoA thioesters of several of these analogs serve as good NMT substrates in vitro, implying that NMT selects fatty acyl substrates primarily on the basis of chain length rather than hydrophobicity. Myristelaidoyl (14:1, delta 9,10-trans) CoA is also a significantly better substrate than myristoleoyl (14:1, delta 9,10-cis) CoA. The fatty acyl group bound to NMT profoundly influences the rate of acylpeptide formation and the affinity of NMT for peptide substrates. However, the peptide substrate bound to NMT does not produce significant alterations in the enzyme's affinity for myristoyl CoA. In vitro characterization of these heteroatom substituted analogs suggests that they will be efficiently incorporated into proteins in vivo and may markedly alter acylprotein targeting and function.

Acylation↗
PubMed1988

Transgenic mice containing intestinal fatty acid-binding protein-human growth hormone fusion genes exhibit correct regional and cell-specific expression of the reporter gene in their small intestine.

The rat intestinal fatty acid binding protein (I-FABP) gene exhibits cell-specific as well as regional differences in its expression within the continuously regenerating small intestinal epithelium. To investigate the underlying mechanisms, we linked portions of its 5' nontranscribed domain to the human growth hormone (hGH) gene and analyzed expression of the hGH reporter in transgenic mice by RNA blot, solution hybridization, and immunocytochemical techniques. Sequences located within 277 nucleotides of the start site of I-FABP transcription are sufficient to limit hGH expression to the intestine. Although the absolute levels of hGH mRNA in the duodenum and proximal jejunum of these transgenic mice were similar to those of I-FABP mRNA, steady-state hGH mRNA concentrations were approximately 100 times lower in their distal small intestine. Addition of nucleotides -278 to -1178 of the I-FABP gene "restored" hGH mRNA concentrations in the distal jejunum and ileum to levels comparable to murine I-FABP mRNA. Serum hGH levels were 1000 times lower in the "short promoter" transgenic mice compared to animals with the "long promoter" transgene, indicating that efficient distal small intestinal hGH expression is required to produce elevated hGH concentrations in serum. The distribution of hGH in villus-associated enterocytes and goblet cells and its lack of expression in the crypts of Lieberkuhn mimicked that of the endogenous I-FABP gene product in all transgenic pedigrees. However, bands of hGH-negative cells extending from the base to the tips of villi were frequently observed in mice that were heterozygous for the short promoter transgene. This mosaic staining was not observed for I-FABP. These data suggest that (i) different cis-acting sequences may be required for complete expression of proximal-distal I-FABP gradients than for recapitulation of its normal crypt-villus tip distribution; (ii) differences may exist in the export pathways of secreted proteins within enterocytes located in various regions of the small intestine; and (iii) there may be subtle genetic differences among various crypt stem cells that can be detected in vivo by observing mosaic patterns of transgene expression along the villus epithelium.

Animals↗
PubMed1988

PARA-SITE: a computer algorithm for rapidly analyzing the physical-chemical properties of amino acid sequences at sites of co- and post-translational protein processing.

A new algorithm is presented which can be used to examine the physical-chemical properties of amino acids at sites of co- or post-translational processing. This algorithm has been incorporated into a computer program known as PARA-SITE. Thirty different parameters can be studied for amino acids which occupy comparable positions in naturally occurring proteins. PARA-SITE should aid in the design and interpretation of protein engineering experiments which seek to dissect structure/activity relationships.

Algorithms↗
PubMed1988

Mechanisms underlying generation of gradients in gene expression within the intestine: an analysis using transgenic mice containing fatty acid binding protein-human growth hormone fusion genes.

The intestine is lined by a continuously regenerating epithelium that maintains gradients in 'liver' fatty acid binding protein (L-FABP) gene expression along its horizontal and vertical axes, i.e., from duodenum to colon and from crypt to villus tip. To identify cis-acting DNA sequences responsible for these regional differences, we linked portions of the L-FABP gene's 5' nontranscribed region to the human growth hormone (hGH) gene and examined hGH expression in transgenic mice. Nucleotides -596 to +21 of the rat L-FABP gene correctly directed hGH expression to enterocytes and hepatocytes. However, anomalous expression was observed in small intestinal crypts, colon, and renal proximal tubular epithelial cells. Addition of nucleotides -4000 to -597 of the L-FABP gene, in either orientation, suppressed renal hGH expression and restored a nearly normal horizontal, but not a vertical, hGH gradient in the intestine. Thus, horizontal gradients of gene expression within the intestine can be maintained by orientation-independent, cis-acting suppressor elements.

Animals↗
PubMed1988

Rat intestinal apolipoprotein B gene expression. Evidence for integrated regulation by bile salt, fatty acid, and phospholipid flux.

We previously reported that intestinal apo B48 synthesis in the rat was unaltered by dietary triglyceride intake but demonstrated regulation in response to biliary lipid availability. Studies are now presented in which the mechanisms underlying biliary lipid dependent expression of intestinal apo B48 synthesis have been investigated further. Bile salt replacement was effective in a dose- and structure-dependent manner in reexpressing intestinal apo B48 synthesis after prolonged bile diversion. Further experiments suggested that this effect of bile salt may be related to facilitated uptake of fatty acid. A role for mucosal phospholipid flux was suggested by studies in which infusion of lysolecithin, with or without Na taurocholate, produced complete reexpression of apo B48 synthesis in jejunal enterocytes. Over a four- to sixfold range of apo B48 synthesis rates in both jejunum and ileum, there was no change in apo B mRNA size or abundance as determined by RNA blot hybridization. Analysis of both intestinal mucosa and microsome lipid content in a variety of settings revealed that apo B48 synthesis rates were correlated with microsome triglyceride fatty acid content (r = 0.65, P less than 0.005) but not free fatty acid or phospholipid content. These studies demonstrate a physiologic role for elements of biliary lipid flux in the regulation of apo B gene expression. The data suggest that an integrated mechanism may exist whereby apo B48 synthesis is related to microsome triglyceride flux, particularly at low levels of lumenal substrate availability.

Animals↗
PubMed1987

The effects of deleting the propeptide from human preproapolipoprotein A-I on co-translational translocation and signal peptidase processing.

The two principal protein components of human high density lipoprotein particles, apoA-I and apoA-II, are initially synthesized as prepropolypeptides. The function of their NH2-terminal prosegments is not known. We have previously shown that deletion of the pentapeptide prosegment (Ala-Leu-Val-Arg-Arg) from human preproapoA-II redirects signal peptidase cleavage to a site located between the second and third residues of the mature protein (Folz, R. J., and Gordon, J. I. (1986) J. Biol. Chem. 261, 14752-14759). The hexapeptide prosegment of human preproapoA-I differs from other NH2-terminal propeptides in that it terminates with paired glutamine residues (Arg-His-Phe-Trp-Gln-Gln). To examine its role in the early events of protein export, we isolated a full-length human preproapoA-I cDNA and deleted its propeptide coding region by oligonucleotide-directed mutagenesis. The effects of this deletion on co-translational translocation and signal peptidase processing were assessed using an in vitro transcription/translation/microsomal membrane processing system. Propeptide deletion reduces the efficiency of co-translational translocation/processing in both reticulocyte and wheat germ lysates but does not affect the fidelity of signal peptidase cleavage. This reduced efficiency does not appear to reflect differences in signal recognition particle-nascent protein interaction as measured by a translational arrest assay. However, differences in translocation/processing rates were noted in a postinitiation translocation/cleavage assay. This assay employed the initiation inhibitor edeine to generate nascent wild type and mutant proteins of increasing chain length which were subsequently presented to canine pancreatic membranes. Unlike preproapoA-I, pre(delta pro)apoA-I retained its ability to undergo translocation and proteolytic processing even after the entire protein was synthesized. These data suggest that the propeptide in human preproapoA-I may play a role in promoting an optimal structure for co-translational translocation and processing.

Amino Acid Sequence↗
PubMed1987

The human and rodent intestinal fatty acid binding protein genes. A comparative analysis of their structure, expression, and linkage relationships.

Intestinal fatty acid binding protein (I-FABP) is believed to participate in the uptake, intracellular metabolism, and/or transport of long chain fatty acids within enterocytes. The 15.1-kDA rodent proteins is a member of a family of low Mr cytoplasmic proteins that have evolved to bind different ligands. We have now determined the nucleotide sequence of the gene encoding human I-FABP and defined the primary structure of its protein product. The human I-FABP gene spans 3382 nucleotides and contains 4 exons (103 or 128, 173, 108, and 312 base pairs). interrupted by 3 introns (1194, 1023, and 444 base pairs). The 132-residue rat and human I-FABPs have 82% amino acid sequence identity. Blot hybridization studies of RNAs prepared from a variety of adult rhesus monkey tissues as well as human intestine and liver indicate that I-FABP mRNA is confined to the intestine. I-FABP mRNA was not detectable in a number of cultured human enterocyte-like cell lines, suggesting it may be a sensitive marker for differentiated, villus-associated, small intestinal lining cells. Given the similar patterns of tissue-specific expression exhibited by the rat and human genes, we compared their 5' nontranscribed regions. Optimal alignments of the two sequences disclosed 64% identity among the 260 nucleotides immediately 5' to the start site of transcription. Matrix plots revealed a 14-nucleotide long repeated sequence (5'-TGAACTTTGAACTT-3') in the 5' nontranscribed region of both genes as well as in a comparable region of another family member that is expressed in enterocytes, cellular retinol binding protein II. The linkage relationships between I-FABP and the homologous liver FABP (L-FABP) gene were defined in mice and humans. The mouse genes were mapped using restriction fragment length polymorphisms and recombinant inbred strains. The I-FABP gene is located on mouse chromosome 3 between the amylase 1,2 (Amy 1,2) and alcohol dehydrogenase 3 (Adh-3) loci while the L-FABP gene is on mouse chromosome 6 within 3 centimorgans of the lymphocyte antigen-2 (Ly-2) locus. Mouse L-FABP may be identical to the major liver protein-1 (Lvp-1) which is encoded by a gene situated within a centimorgan of Ly-2. Human gene mapping studies were carried out using a panel of mouse-human somatic cell hybrid clones as well as in situ hybridization to metaphase chromosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1987

Crystallization of rat cellular retinol binding protein II. Preliminary X-ray data obtained from the apoprotein expressed in Escherichia coli.

Rat cellular retinol-binding protein II (CRBP II) is a member of a family of cytoplasmic proteins which bind hydrophobic ligands. CRBP II is thought to participate in the intestinal absorption and intracellular metabolism of retinoids. We have previously described the crystallization of a homologous rat intestinal fatty acid-binding protein (I-FABP) isolated from Escherichia coli containing a suitably constructed prokaryotic expression vector (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J., J. Biol. Chem. 262, 5428-5430). We have now efficiently expressed rat CRBP II in E. coli. The E. coli-derived protein, which does not contain any bound retinoid, has been purified and crystals grown from solutions of polyethylene glycol 4000. Crystals of apo-CRBP II are triclinic, space group P1, a = 36.8 A, b = 64.0 A, c = 30.4 A; alpha = 92.8 degrees, beta = 113.5 degrees, gamma = 90.1 degrees. Each unit cell contains two molecules of the 134-residue apoprotein. X-ray diffraction data suggest that the unit cell parameters of crystalline apo-CRBP II resemble those of I-FABP. Comparison of the tertiary structures of E. coli-derived rat I-FABP and CRBP II should provide insights about how these proteins evolved to bind different hydrophobic ligands.

Animals↗
PubMed1987

Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli.

Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.

Amino Acids↗
PubMed1987

Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta.

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.

Algorithms↗