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J I Gordon

Publications and source records attributed to J I Gordon.

At least 163 records · Page 9Linked to original sources
PubMed1991

An analog of myristic acid with selective toxicity for African trypanosomes.

Trypanosoma brucei, the protozoan parasite responsible for African sleeping sickness, evades the host immune response through the process of antigenic variation. The variant antigen, known as the variant surface glycoprotein (VSG), is anchored to the cell surface by a glycosyl phosphatidylinositol (GPI) structure that contains myristate (n-tetradecanoate) as its only fatty acid component. The utilization of heteroatom-containing analogs of myristate was studied both in a cell-free system and in vivo. Results indicated that the specificity of fatty acid incorporation depends on chain length rather than on hydrophobicity. One analog, 10-(propoxy)decanoic acid, was highly toxic to trypanosomes in culture although it is nontoxic to mammalian cells.

Acyl Coenzyme A↗
PubMed1991

Characterization of the peripheral neuropathy in neonatal and adult mice that are homozygous for the fatty liver dystrophy (fld) mutation.

In a previous report (Langner, C. A., Birkenmeier, E. H., Ben-Zeev, O., Schotz, M. C., Sweet, H. O., Davisson, M. T., and Gordon, J. I. (1989) J. Biol. Chem. 264, 7994-8003), we characterized the early developmental phenotype of mice that were homozygous for the autosomal recessive fatty liver dystrophy (fld) mutation. Shortly after birth, these mice can be distinguished from their +/? littermates by large pale livers, hypertriglyceridemia, elevations in hepatic apolipoprotein A-IV and apoC-II mRNA levels, and tissue-specific decreases in lipoprotein lipase and hepatic lipase activities. These traits resolve by the early weaning period. We have now characterized a second feature of this mutation: a peripheral neuropathy that becomes manifest by an abnormal gait at the end of the second postnatal week and persists through adulthood. Electron microscopic studies of sciatic nerves from 4-day-to 1-year-old fld/fld mice demonstrated a variety of abnormalities including thin, poorly compacted myelin sheaths, active myelin breakdown, and enlarged Schwann cell mitochondria and nuclei. Western blot analysis of sciatic nerve homogenates prepared from 1 to 3-month-old fld/fld mice and their +/? littermates indicated that homozygous animals have striking reductions in two peripheral nerve myelin-associated proteins, P0 and P2. The steady-state level of apoE, a protein induced during nerve regeneration, is markedly elevated. Furthermore, two axon-specific proteins, neurofilament 68K and growth-associated 43 protein, display altered expression in adult fld/fld sciatic nerves. High performance thin-layer chromatography revealed deficiencies in phospholipids, glycosphingolipids, and some neutral lipids in fld/fld sciatic nerves harvested during the first several months of life (compared to their +/? littermates). Cholesterol esters were elevated in homozygotes. By contrast, no differences in brain lipids were noted between fld/fld animals and their +/? littermates. These data suggest that the fld mutation is associated with an abnormality of myelin formation (dysmyelination) as well as demyelination and axonal degeneration that persists despite apparent resolution of the neonatal hypertriglyceridemia and associated lipase abnormalities. These findings establish the fld/fld mouse as an excellent model system for analyzing homeostatic mechanisms that modulate lipid metabolism in newborn mice and for examining the pathogenesis of peripheral neuropathies associated with dyslipidemias.

Aging↗
PubMed1991

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli.

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.

Acyltransferases↗
PubMed1991

Kinetic and structural evidence for a sequential ordered Bi Bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The mechanism of catalysis of Escherichia coli-derived Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase (NMT) has been characterized. Previous studies indicated that a high affinity reaction intermediate forms between NMT and myristoyl-CoA in the absence of a peptide substrate. This complex has been further characterized using S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog. Binding studies involving this analog, as well as myristoylpeptide and CoA, have indicated that the CoA moiety of the acyl substrate is retained in the acyl-NMT complex prior to peptide addition. These structural data, along with kinetic studies of myristoylpeptide and CoA product inhibition, indicate that the mechanism of catalysis of NMT is ordered Bi Bi, with myristoyl-CoA binding to NMT occurring prior to peptide binding and CoA release taking place before release of acyl peptide. Further analyses of the interactions between NMT, acyl peptide, and CoA demonstrate that NMT is able to deacylate a myristoylpeptide in the presence of CoA.

Acyltransferases↗
PubMed1991

Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocyte and enteroendocrine cell populations of fetal transgenic mice.

The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations.

Animals↗
PubMed1991

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.

Antiviral Agents↗
PubMed1991

Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma.

Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P. O., Devine, C.S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S.A. 87, 1506-1510). Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose. Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus. Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid. The second form of the protein was not myristoylated. Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits. The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions. Thus, myristoylation increases the affinity of alpha subunits for beta gamma. We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.

Acylation↗
PubMed1991

Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.

Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.

Animals↗
PubMed1991

Temporal and spatial patterns of transgene expression in aging adult mice provide insights about the origins, organization, and differentiation of the intestinal epithelium.

We have used liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to explore the temporal and spatial differentiation of intestinal epithelial cells in 1- to 12-month-old transgenic mice. The intact, endogenous L-FABP gene (Fabpl) was not expressed in the colon at any time. Young adult transgenic mice containing nucleotides -596 to +21 of the rat L-FABP gene linked to the hGH gene (minus its 5' nontranscribed domain) demonstrated inappropriate expression of hGH in enterocytes and many enteroendocrine cells of most proximal and mid-colonic crypts (glands). Rare patches of hGH-negative crypts were present. With increasing age, a wave of "extinction" of L-FABP (-596 to +21)/hGH expression occurred, first in the distal colon and then in successively more proximal regions, leaving by 10 months of age only rare hGH-positive multicrypt patches. At no time during this progressive silencing of transgene expression were crypts observed that contained a mixture of hGH-positive and -negative cells at a particular cell stratum. Young (5-7 weeks) mice containing a L-FABP (-4000 to +21)/hGH transgene also demonstrated inappropriate expression of the transgene in most proximal colonic crypts. However, the additional 3.3 kilobases of upstream sequence resulted in much more rapid extinction of reporter expression, leaving by 5 months of age only scattered single crypts with detectable levels of hGH. This age-related extinction of L-FABP/hGH expression did not involve enterocytes and enteroendocrine cells in the (proximal) small intestine. These results indicate that cis-acting elements outside of nucleotides -4000 to +21 are necessary to fully modulate suppression of colonic L-FABP expression. They also define fundamental changes in colonic epithelial cell populations during adult life. Our data suggest that (i) a single stem cell gives rise to all cells that populate a given colonic crypt, (ii) stem cells represented in several adjacent crypts may be derived from a common progenitor, and (iii) such a progenitor cell may repopulate colonic crypts with stem cells during adult life. Since each colonic crypt contains the amplified descendants of its stem cell, transgenes may be powerful tools for characterizing the spatial and biological features of gut stem cells and their progenitors during life.

Aging↗
PubMed1991

Epithelial cell differentiation in normal and transgenic mouse intestinal isografts.

Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

Acyltransferases↗
PubMed1991

Expression of a liver fatty acid binding protein/human decay-accelerating factor/HLA-B44 chimeric gene in transgenic mice.

The intestinal epithelium is characterized by the rapid and continuous renewal of its four principal cell types and by its ability to establish and maintain remarkably complex spatial differentiation along its crypt-to-villus and duodenal-to-colonic axes. We have previously used transgenic mice containing liver fatty acid binding protein/human growth hormone (L-FABP/hGH) fusion genes to analyze the molecular mechanisms responsible for encoding positional information in this epithelium. Because these studies could not distinguish whether cis-acting sequences in the L-FABP promoter or hGH structural gene were responsible for the observed cellular and regional patterns of transgene transcription in the gut, a second model fusion gene has now been constructed. It consists of nucleotides -596 to +21 of rat L-FABP linked to a cDNA encoding a chimeric protein, human decay-accelerating factor (DAF, minus the site of attachment of its COOH-terminal glycophospholipid anchor), coupled to the transmembrane (TM) and cytoplasmic domains of human HLA-B44. RNA blot hybridization and immunocytochemical analyses revealed that the cell-specific and region-specific expressions of DAF-TM and hGH in adult mice appear identical along both axes of the gut, indicating that cis-acting elements contained within the 5' nontranscribed region of the L-FABP gene rather than in the reporter are largely responsible for these observed patterns of transgene expression. Unlike pre-hGH, a prototypical secreted protein, DAF-TM is a membrane protein. The ability to direct its expression along the length of both axes of the gut provides an opportunity to analyze in vivo the sorting pathways of membrane-associated proteins in normal epithelial cells as a function of their location and differentiation. Light microscopic studies indicate that DAF-TM is targeted to the basolateral and apical surfaces of villus-associated enterocytes.

Amino Acid Sequence↗
PubMed1990

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro.

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.

Amino Acid Sequence↗
PubMed1990

Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium.

Enterocytes located in the pig distal small intestine (ileum) contain a cytosolic protein that is homologous to two proteins that are also synthesized in these cells: intestinal and "liver" fatty acid-binding proteins (I- and L-FABPc, respectively). To begin to investigate the functional interrelationships of these three proteins, we compared their patterns of tissue-specific expression and developmental regulation in the mouse. Blot hybridization analyses of RNA prepared from 12 adult tissues revealed that this mRNA was confined to the small intestine. Unlike I- and L-FABPc mRNA, which are most abundant in the proximal jejunum, this mRNA is most abundant in the ileum. While I- and L-FABPc gene transcription commence in late fetal life coincident with initial cytodifferentiation of the mouse gut epithelium, the ileal gene is activated later, at the suckling/weaning transition (postnatal day 12). The ileal location and developmental pattern of expression suggested that this protein may play a role in the intracellular transport of bile salts in the ileal epithelium. To test this hypothesis, we expressed the porcine ileal peptide (PIP) in Escherichia coli, purified it to apparent homogeneity, and analyzed its binding properties for bile acids and fatty acids using 13C NMR spectroscopy. Like I-FABPc, PIP binds palmitate and oleate with a 1:1 molar stoichiometry. However, unlike I-FABPc PIP binds chenodeoxycholate. In addition, the presence of chenodeoxycholate blocks fatty acid binding to PIP, but not to I-FABPc. E. coli-derived PIP was subsequently crystallized with and without chenodeoxycholic acid. All crystals are orthorhombic in the P2(1)2(1)2(1) space group. The unit cell dimensions are a = 36.15 A, b = 50.13 A, and c = 67.18 A.

Aging↗
PubMed1990

Folding of a predominantly beta-structure protein: rat intestinal fatty acid binding protein.

The equilibrium and kinetic properties of the unfolding-refolding transitions of Escherichia coli derived rat intestinal fatty acid binding protein have been examined using several different denaturants. This protein, which contains 2 tryptophans but no prolines or cysteines, has a predominantly beta-structure: its 10 antiparallel beta-strands are organized into 2 orthogonal sheets surrounding a large solvent-filled internal cavity. For urea and guanidine hydrochloride, the completely reversible transition was monitored by circular dichroism, absorbance, and fluorescence spectroscopy. Each of these data sets was best fit by a simple, two-state model involving only native and unfolded forms. However, linear extrapolation to determine the free energy of folding in the absence of denaturant resulted in different values for the free energy of folding depending upon which denaturant was used. When fluorescence was used to monitor the transition, the extrapolated free energy estimates for the two denaturants were markedly different: 10.03 +/- 0.24 kcal mol-1 for urea versus 5.22 +/- 0.33 kcal mol-1 for guanidine hydrochloride. The midpoints of these transitions were 5.51 and 1.36 M, respectively. The transition caused by either denaturant as monitored by circular dichroism and absorbance spectroscopy was virtually coincident with that monitored by fluorescence, further supporting the assignment of a two-state model for the equilibrium results. The addition of a 2-fold molar excess of ligand (oleate) increased the extrapolated estimates approximately 2.5 kcal mol-1 for both denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase.

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.

Amino Acid Sequence↗
PubMed1990

Compartmentalization of mammalian proteins produced in Escherichia coli.

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.

Amino Acid Sequence↗