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J C Wallace

Publications and source records attributed to J C Wallace.

At least 73 records · Page 4Linked to original sources
PubMed1996

The insulin-like growth factor (IGF) binding site of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) probed by iodination.

The insulin-like growth factor (IGF) binding site of bovine insulin-like growth factor binding protein 2 (bIGFBP-2) has been probed by chemical iodination. Tyrosyl residues of bIGFBP-2 were reacted by chloramine T-mediated iodination. The modification patterns of free bIGFBP-2 and bIGFBP-2 associated with insulin-like growth factor II (IGF-II) were compared by tryptic mapping using electrospray mass spectrometry and N-terminal sequencing. The presence of bound IGF-II resulted in protection of tyrosine at position 60 from iodination measured by the relative loss of tyrosine specific fluorescence and the incorporation of the radioisotope 125I. In addition, the pattern of iodine incorporation of bIGFBP-2 was not different whether IGF-I or IGF-II was the protective ligand. bIGFBP-2, when iodinated alone sustained a 8-fold loss of binding affinity for IGF-I and a 4-fold loss in binding affinity for IGF-II. In contrast, bIGFBP-2 iodinated while complexed with either IGF-I or IGF-II retained the same binding affinity for IGF-I or IGF-II as non-iodinated bIGFBP-2. We conclude that tyrosine 60 lies either in a region of bIGFBP-2 which directly interacts with both IGF-I and IGF-II or lies in a region of bIGFBP-2 which undergoes a conformational change that is important for IGF binding. Furthermore, iodination of tyrosine residues at positions 71, 98, 213, 226, and 269 has no detectable impact on binding of bIGFBP-2 to the IGFs.

Amino Acid Sequence↗
PubMed1996

Identification of novel alternatively spliced pyruvate carboxylase mRNAs with divergent 5'-untranslated regions which are expressed in a tissue-specific manner.

We have identified and characterized multiple mRNA transcripts of rat and human pyruvate carboxylases [EC 6.4.1.1] using rapid amplification of cDNA 5' ends-polymerase chain reaction (RACE-PCR). Five alternative forms of rat pyruvate carboxylase cDNAs have been identified in liver, kidney, brain, and adipose tissue and these are expressed in a tissue-specific manner. Two alternative forms of human pyruvate carboxylase cDNA have also been identified in liver. These pyruvate carboxylase cDNAs have a common coding region but differ in their 5' untranslated regions (5'UTRs), suggesting that they are generated by alternative splicing of the primary transcript. Southern blot analysis of restriction enzyme digested rat genomic DNA revealed that pyruvate carboxylase is encoded by a single copy gene.

Alternative Splicing↗
PubMed1996

Cloning, sequencing and expression of rat liver pyruvate carboxylase.

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated M(r) of 129848. This is flanked by a 5' untranslated region of 66 bp and a 3' untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249-10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

Amino Acid Sequence↗
PubMed1996

Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I.

Des(1-3)IGF-I, a truncated variant of human IGF-I with the tripeptide Gly-Pro-Glu absent from the N-terminus, has been isolated from bovine colostrum, human brain and porcine uterus. This protein probably results from post-translational cleavage of IGF-I. Des(1-3)IGF-I generally is about 10-fold more potent than IGF-I at stimulating hypertrophy and proliferation of cultured cells, a consequence of much reduced binding to IGF-binding proteins, in turn caused by the absence of the glutamate at position 3. The increased potency is retained in part when the variant is administered in vivo, with selective anabolic effects particularly evident in gut tissues. Clinical opportunities for des(1-3)IGF-I have not yet been evaluated, but could apply in catabolic states as well as for the treatment of inflammatory bowel diseases.

Amino Acid Sequence↗
PubMed1996

Production of a human epidermal growth factor fusion protein and its degradation in rat gastrointestinal flushings.

This study describes the biosynthesis of a human epidermal growth factor fusion protein, Long EGF, that has a 53 amino acid extension peptide derived from the 46 N-terminal amino acids of porcine GH. The approach allowed the production of Long EGF at high efficiency due to the expression of the fusion protein in high yield as inclusion bodies in Escherichia coli. Long EGF had a slightly lower potency compared with native EGF in a range of assays, including binding to anti-EGF antibodies or the EGF receptor, stimulation of Balb/3T3 fibroblast and rat intestinal epithelial cell growth, as well as counteracting the inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta 1. Degradation of Long EGF and native EGF was compared in gastrointestinal flushings as an indication of whether the EGF domain of the fusion protein would be protected from proteolytic cleavage and be useful as a trophic agent in the gut. Incubation with flushings from the stomach or jejunum of rats caused rapid cleavage of the extension peptide, releasing native EGF. A C-terminal truncation of Arg53 in the stomach and a removal of the C-terminal pentapeptide (49 Trp-Trp-Glu-Leu-Arg53) in the small bowel was demonstrated by N-terminal sequencing and mass spectrometry. The degradation patterns were reflected by changes in migration of products on SDS-PAGE and in subsequent binding activities to the EGF receptor and anti-EGF antibodies. The data show that a human EGF fusion protein can be produced efficiently in a bacterial expression system and that it retains biological activity in vitro. Although the extension peptide was rapidly cleaved from Long EGF in both stomach and small bowel producing similar biological activity to native EGF, it could not prevent subsequent degradation of the EGF domain. Other strategies are being investigated to develop an effective oral form of EGF that resists digestion by proteases in the gastrointestinal tract.

3T3 Cells↗
PubMed1996

Insulin-like growth factor (IGF)-I A- and B-domain analogues with altered type 1 IGF and insulin receptor binding specificities.

Insulin-like growth factor-I (IGF-I) analogues were produced with the aim of identifying IGF-I residues that contribute to the specificity of binding to the type 1 IGF receptor as opposed to the insulin receptor. Receptor binding properties of a series of A- and B-domain analogues were compared using rat L6 myoblasts, soluble human IGF type 1 receptors and soluble human insulin receptor isoforms HIR-A (-Ex11) and HIR-B (+Ex11). IGF-I analogues, [Leu8] IGF-I and [Phe59] IGF-I, were shown to exhibit respectively, a 28- and 17-fold decrease in affinity for the HIR-A with only a 6- and 5-fold decrease in affinity for the human IGF type 1 receptor. In contrast, the analogue [His4] IGF-I was equipotent to IGF-I in binding to the soluble type 1 IGF receptor while showing 7-fold and 4-fold increases in HIR-A and HIR-B binding respectively. Furthermore, [Leu62] IGF-I was 8-fold less potent than IGF-I in soluble IGF type 1 receptor binding but only showed a 2-fold decrease in HIR-A and HIR-B binding. Our study supports the conclusion that the co-evolution of the IGF-I and insulin receptor/ligand systems has resulted in subtle structural differences in the A- and B-regions of each ligand important for defining receptor binding specificity.

3T3 Cells↗
PubMed1996

Design and characterisation of long-R3-insulin-like growth factor-I muteins which show resistance to pepsin digestion.

Site-directed mutagenesis was used to construct pepsin-resistant, single-point mutations of the N-terminal extended IGF-I analogue, long-R3-IGF-I. In order to identify the most susceptible sites, the kinetics of long-R3-IGF-I digestion by purified porcine pepsin were determined. Pepsin initially cleaved the Leu10-Phe11 bond in the N-terminal extension peptide to generate FVN-R3-IGF-I, followed in rapid succession by cleavage at Gln15-Phe16, Tyr24-Phe25, Leu10-Val11 and Met59-Tyr60 in the IGF-I moiety. Single-point mutations at these sites were designed on the basis of the preferred cleavage bonds for pepsin, as well as amino acid substitutions less likely to disturb protein structure. These included Leu10Val, Phe16Ala, Phe25Leu, Asp53Glu and Met59Gln. All five muteins retained growth-promoting activity equivalent to or higher than that of IGF-I. In terms of pepsin susceptibility, Leu10Val and Asp53Glu were degraded as rapidly as the parent long-R3-IGF-I, Met59Gln and Phe25Leu were partially stabilised, and Phe16Ala showed a marked improvement in stability over a wide range of pepsin:substrate ratios. Accordingly, the Phe16Ala mutein, long-R3A16-IGF-I, has potential for oral applications to enhance gastric growth and repair.

Amino Acid Sequence↗
PubMed1996

Radiation-induced formation of a crosslink between base moieties of deoxyguanosine and thymidine in deoxygenated solutions of d(CpGpTpA).

A new type of tandem base lesion has been observed in d(CpGpTpA) X-irradiated in aqueous solution. The lesion is attributed to the formation of a covalent bond between the C8 carbon atom of guanine and the methyl carbon atom of thymine. This tandem base lesion is formed in the absence of oxygen. It is the main product produced by ionizing radiation under these conditions.

Acid Phosphatase↗
PubMed1995

Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation.

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3' end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.

Amino Acid Sequence↗
PubMed1995

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae.

We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae. The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues (M(r) 76.4 kDa) with strong sequence similarities to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae.

Alleles↗
PubMed1995

Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties.

The oxidative folding of human insulin-like growth factor (IGF)-I yields two major disulphide folding isomers. In the present study, B-domain analogues of IGF-I were used to investigate the effect of mutations on the folding reaction and to investigate the functional implications of misfolding. The analogues used were substitutions of the native Glu3 by Gly or Arg, or the native Glu9 by Lys. IGF-I and these analogues were also prepared attached to a hydrophobic 13-amino-acid N-terminal extension, Met-Phe-Pro-Ala-Met-Pro-Leu-Ser-Ser-Leu-Phe-Val-Asn, referred to as 'Long-IGF-I' analogues. Each IGF was fully reduced and refolded to yield native and misfolded isomers, which were subsequently purified for biological characterization. Analysis of the folding reaction at equilibrium revealed a distribution of folding isomers characteristic for each peptide. The yield of the native disulphide folding isomer was increased for the Glu3 substitutions, but not for the Glu9 substitution. The main alternative folding isomer was present in the IGF-I analogues in reduced proportions. Except for [Gly3]IGF-I the N-terminal extension increased the yield of the native isomer which was maximal for the analogue Long-[Arg3]IGF-I. A folding intermediate for the latter analogue was isolated and partially characterized. The biological assays showed that all the main alternative isomers bound poorly to IGF-binding proteins (IGFBPs) secreted by L6 myoblasts. Moreover, these isomers bound to the type 1 IGF receptor with 0.5-25% the affinity of the native isomer. In a rat L6 myoblast protein-synthesis assay, the observed biological activity of the native and main alternative isomers was explained by their modified IGFBP- and receptor-binding properties. We propose that the N-terminal extension imparts a steric constraint at a crucial point in folding, thus allowing native disulphide bonds to form efficiently.

Amino Acid Sequence↗
PubMed1995

Solution structure of a nucleic acid photoproduct of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT) determined by NMR and restrained molecular dynamics: structural comparison of two sequence isomer photoadducts (d-U5p5T and d-T5p5U).

Acetone-sensitized irradiation using UV-B (sun lamp, lambda max = 313 nm) of deoxyfluorouridylyl-(3'-5')-thymidine monophosphate (d-FpT, F = fluorouracil), produces two major photoproducts, the cis-syn cyclobutane-type photodimer and a defluorinated (5-5) photoadduct, d-U5p5T. Product distribution is dependent on the pH of the irradiation solution, as was the case of irradiated d-TpF. At high pH (8-10) the (5-5) photoadduct is the major photoproduct. Irradiation of d-FpT shows a much faster photodegradation rate than the sequence isomer d-TpF. Multinuclear NMR experiments establish the formation of (5-5) covalent bonding between the C5 (d-U5p-, where the fluorine had been) and the C5 (-p5T) and the C6 (-p5T) acquires an OH group. NOE interproton distances and dihedral angles derived from J coupling analysis are constrained to refine model structures of d-U5p5T in restrained molecular dynamics calculations. The resultant structures obtained show 5S-6S as the most chiralities of the C5 and C6 atoms of the thymine, which is the opposite chirality to the corresponding atoms in the sequence isomer d-T5p5U. The orientation of the C5 substituents (-p5T fragment), the CH3 and the uracil are pseudo-axial and pseudo-equatorial respectively. Glycosidic angles are in the anti regions for both the d-U5p- and -p5T residues. Averaged backbone conformations of the two photoadducts, d-U5p5T and d-T5p5U, are similar, although the overall structure of d-U5p5T appears much more flexible than that of d-T5p5U. In particular, the sugar conformations of the 5'-end residues show a remarkable difference in flexibility.

DNA Damage↗
PubMed1995

Solution structure of human insulin-like growth factor II. Relationship to receptor and binding protein interactions.

The three-dimensional structure of human insulin-like growth factor (IGF) II in aqueous solution at pH 3.1 and 300 K has been determined from nuclear magnetic resonance data and restrained molecular dynamics calculations. Structural constraints consisting of 502 NOE-derived distance constraints, 11 dihedral angle restraints, and three disulfide bridges were used as input for distance geometry calculations in DIANA and X-PLOR, followed by simulated annealing refinement and energy minimization in X-PLOR. The resulting family of 20 structures was well defined in the regions of residues 5 to 28 and 41 to 62, with an average pairwise root-mean-square deviation of 1.24 A for the backbone heavy-atoms (N, C2, C) and 1.90 A for all heavy atoms. The poorly defined regions consist of the N and C termini, part of the B-domain, and the C-domain loop. Resonances from these regions of the protein gave stronger cross peaks in two dimensional NMR spectra, consistent with significant motional averaging. The main secondary structure elements in IGF-II are alpha-helices encompassing residues 11 to 21, 42 to 49 and 53 to 59. A small anti-parallel beta-sheet is formed by residues 59 to 61 and 25 to 27, while residues 26 to 28 appear to participate in intermolecular beta-sheet formation. The structure of IGF-II in the well-defined regions is very similar to those of the corresponding regions of insulin and IGF-I. Significant differences between IGF-II and IGF-I occur near the start of the third helix, in a region known to modulate affinity for the type 2 IGF receptor, and at the C terminus. The IGF II structure is discussed in relation to its binding sites for the insulin and IGF receptors and the IGF binding proteins.

Amino Acid Sequence↗
PubMed1995

Analysis of insulin-like growth factor binding proteins in the tammar wallaby, Macropus eugenii.

The insulin-like growth factors (IGFs) are important regulators of growth and development in eutherian mammals. In this study we have analyzed circulating insulin-like growth factor binding proteins (IGFBPs) by Western ligand blotting (WLB) and neutral gel filtration, and hepatic IGFBP mRNA transcripts in the wallaby, Macropus eugenii, a marsupial in which the fetus is born at an immature stage compared to eutherian mammals. Plasma from male and female adults, lactating mothers, and pouch young contains an IGF binding species consisting of a 42- to 50-kDa doublet, 30, 28, 24, and approximately 200 kDa, as shown by WLB. This pattern of IGFBPs is very similar to that observed in human and sheep. Neutral gel filtration revealed IGF binding activity of molecular size 70-160 kDa. When electrophoresed under nonreducing, denaturing conditions, the 70- to 160-kDa IGF binding activity appeared as a 42- to 50-kDa doublet, indicating the presence of a high-molecular-weight circulating IGF binding complex. These properties are again analogous to those observed for eutherian IGFBPs. Northern analysis of total wallaby liver RNA detected transcripts of 1.8 kb for IGFBP-1 and 1.6 kb for IGFBP-2, the same as those observed in the sheep and rat. Transcripts of 3.2 kb were detected for IGFBP-4, larger than the major band of 2.6 kb observed for the sheep and rat. Transcripts representing wallaby IGFBP-3 mRNA were not detected using a rat IGFBP-3 cDNA probe. Circulating IGFBPs were analyzed in plasma from wallaby mothers and their pouch young. There were no changes in circulating IGFBPs in the wallaby mothers throughout lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Radiation chemistry of d(ApCpGpT).

The radiation chemistry of the DNA tetranucleoside triphosphate d(ApCpGpT) was investigated. Irradiations were carried out on aqueous solutions saturated with oxygen (with and without added Cu++), nitrogen or nitrous oxide. When oxygen was present, principal products were formed by hydroxylation at the 8-position of guanine and by degradation of thymine leaving a formamido remnant. Products were also formed containing both of the aforementioned lesions at adjacent deoxyguanosine and pyrimidine nucleosides. Other products resulted from rearrangement of the thymine ring generating two diastereoisomers of the 5-methyl-5-hydroxyhydantoin modification of d(ApCpGpT). Rearrangement of the cytosine ring occurred generating imidazolidine products and a hydantoin product. The product profiles are similar when either an N2O or N2 gaseous environment is maintained. However, in the latter case the dihydrothymine modifications of d(ApCpGpT) are markedly enhanced. Other products include an 8,5' cyclized product formed from the 2'-deoxyadenosine nucleoside and the 8-hydroxyguanine modification. 6-Hydroxy-5,6-dihydrothymine and 5,6-dihydroxy-5,6-dihydrothymine modifications of the thymidine nucleoside were also observed. A strand break product formed in oxygenated solution is also produced in nitrous oxide saturated solutions. Scission of the deoxyadenosine terminus was also observed. The effect of several of these lesions on d(ApCpGpT) as substrate for nuclease P1, bovine spleen phosphodiesterase and snake venom phosphodiesterase was studied.

Oligodeoxyribonucleotides↗
PubMed1995

Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli.

Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.

Amino Acid Sequence↗