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W J Rutter

Publications and source records attributed to W J Rutter.

At least 37 records · Page 2Linked to original sources
PubMed1994

Exogenous acetate reconstitutes the enzymatic activity of trypsin Asp189Ser.

The specificity of trypsin for Arg- and Lys-containing substrates depends upon the presence of Asp189 at the base of the primary binding pocket. The crystal structure of anionic rat trypsin D189S complexed with BPTI reveals that removal of the aspartate side chain permits the binding of a well-ordered acetate ion in a similar position. The acetate makes polar interactions with Gly226, Tyr228, and several water molecules and is further accommodated by rotation of the Ser189 side chain out of the binding pocket. The carboxylate group of the acetate anion is oriented toward the substrate in a manner similar to that of Asp189 and Asp226 in wild-type trypsin and trypsin D189G/G226D. Evaluation of kinetic parameters for amide substrate cleavage by trypsin D189S shows that high concentrations of acetate increase the catalytic efficiency of the enzyme by as much as 300-fold. Under these conditions, the rate of substrate turnover toward a peptidylarginine amide substrate equals that of wild-type trypsin. These data demonstrate that the well-established requirement for a negatively charged moiety at the base of the trypsin specificity pocket may be fulfilled by a noncovalently bound ligand. The binding pocket of this variant maintains a trypsin-like conformation, explaining the inability of the mutant enzyme to efficiently hydrolyze chymotrypsin substrates possessing Phe in the P1 position.

Acetates↗
PubMed1994

Purification of E. coli-synthesized Pan proteins and development of a Pan-specific monoclonal antibody.

The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan-2 (E12), are produced by the mechanism of alternative transcript splicing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification scheme was developed. Purified Pan-2 was used to immunize Smith-Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2. Deletion mapping and sequence transfer studies have localized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2. This region is conserved in Pan-1 and Pan-2. The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta. By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.

Animals↗
PubMed1993

Association and phosphorylation-dependent dissociation of proteins in the insulin receptor complex.

Receptor tyrosine kinases have been found to interact with a variety of specific signaling molecules. To detect molecules that interact with the insulin receptor, we have produced a modified insulin receptor with an additional epitope allowing rapid purification under mild conditions of the insulin receptor complex. By this method we have found multiple proteins (including the p85 subunit of phosphatidylinositol 3'-kinase and the ras GTPase-activating protein) that specifically associate with the activated (phosphorylated) insulin receptor (insulin receptor complex proteins) but are released from the complex after they are phosphorylated on tyrosine residues. We have also shown that tyrosine phosphorylation of p85 by the activated insulin receptor blocks binding to the activated receptor. These results suggest that association of proteins with the insulin receptor complex is controlled by phosphorylation of the receptor, while dissociation of insulin receptor complex proteins is controlled in turn by phosphorylation of the proteins in the insulin receptor complex. This process results in the dispersion of phosphorylated insulin receptor complex proteins into the cell.

Base Sequence↗
PubMed1993

Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases.

The X protein of hepatitis B virus (HBV-X) can act as a transactivator of transcription but its mechanism of action remains obscure. We have analyzed HBV-X transactivation in several cell types using 13 unrelated viral and cellular promoters and found that transactivation is more or less apparent in most cell types and is promiscuous and unrelated to specific sequence motifs within the target promoters. In general, though, HBV-X appears to act on enhancer elements since HBV-X had no effect on a minimal promoter, whereas HBV-X was able to transactivate after insertion of an AP-1 minienhancer. Several lines of evidence exclude the possibility that HBV-X interacts directly with the AP-1 enhancer or its binding proteins and suggest that the proximal target of HBV-X is peripheral to the transcription complex. This hypothesis is supported by the observation that inhibition of serine/threonine kinases, which regulate AP-1 activity (phorbol ester down-regulation or staurosporine inhibition of protein kinase C and a dominant negative mutant of Raf-1), blocked the ability of HBV-X to transactivate without affecting basal promoter activity. Furthermore, basal transcription from the AP-1-dependent promoter was increased by overexpression of protein kinase C and Raf-1 but HBV-X was unable to further stimulate, indicating that these kinases act subsequently to HBV-X. These data suggest that transactivation by HBV-X is an indirect result of the activation of cellular serine/threonine kinases including protein kinase C and Raf-1. This mode of action implies that HBV-X may affect other cellular processes, besides transcription, that are regulated by these kinases.

Animals↗
PubMed1993

A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain.

We have identified a breast carcinoma tyrosine phosphoprotein, discoidin domain receptor (DDR), that defines an unusual class of receptor tyrosine kinases. The DDR cDNA predicts a C-terminal tyrosine kinase domain and an N-terminal domain similar to the Dictyostelium discoideum lectin discoidin I. These domains are connected by an extraordinary hydrophilic proline/glycine-rich domain, which is interrupted by a predicted transmembrane sequence. This extended proline/glycine-rich region may be required for an unusual geometry of interaction with ligand or substrates. Discoidin I domains are also found in other proteins, including coagulation factors V and VIII, and may represent a class of domains that interact with specific cell surface molecules.

Amino Acid Sequence↗
PubMed1993

Analysis of enzyme specificity by multiple substrate kinetics.

Multiple approaches for screening large sets of compounds for a specific function are of growing interest. The use of substrate mixtures to characterize the specificity of enzymes has been limited so far to compounds with similar kinetic parameters, because the data were analyzed by applying the kinetics of two competing substrates. In this study we introduce a statistical method for the analysis of reactions with many competing substrates which makes use of the specific features of multiple substrate kinetics. It is assumed that the relative concentrations of all substrates in a mixture can be monitored by high-performance liquid chromatography or a similar technique. Relative second-order rate constants, i.e., kcat/KM values, can be calculated for all substrates in the mixture from the resulting data set. The calculation uses the fact that there is a relationship between the concentrations of all pairs of substrates in the mixture. As a result, the precision of the calculated parameters is increased and the range of kinetic constants that can be obtained from one experiment is considerably expanded. Simulations demonstrate that the precision in the kinetic parameters increases with the number of substrates in the mixture. In fact, estimation of ratios of rate constants can be improved (or made possible) for substrates with order of magnitude differences in reactivity by adding "dummy" substrates with intermediate reactivities, even though the rate constants for dummy substrates are themselves of no intrinsic interest.

Binding, Competitive↗
PubMed1993

Mapping the S' subsites of serine proteases using acyl transfer to mixtures of peptide nucleophiles.

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.

Acyltransferases↗
PubMed1993

Initiation of gastrin expression during the development of the mouse pancreas.

Gastrin expression occurs in the pancreas in only two situations: (1) in cases of gastrinoma and (2) in the embryonic/fetal pancreas. The initiation of gastrin expression in the embryonic pancreas may be recapitulated during gastrinoma tumorigenesis. For this reason, we have tried to identify the point of onset of gastrin expression in the developing pancreas. Previously, determining the point of onset for genes in embryonic tissues has been difficult because of low expression levels and small tissue samples. Using the sensitive polymerase chain reaction assay, we were able to determine, with a sensitivity of 10 molecules of mRNA, the earliest expression of gastrin in the developing pancreas. This expression occurred at 30 somites, or at a gestation of 9.5 to 10 days.

Animals↗
PubMed1993

Modulating effects of the extracellular sequence of the human insulinlike growth factor I receptor on its transforming and tumorigenic potential.

We reported previously that an N-terminally truncated insulinlike growth factor I receptor (IGFR) fused to avian sarcoma virus UR2 gag p19 had a greater transforming potential than did the native IGFR, but it failed to cause tumors in vivo. To investigate whether the 36 amino acids (aa) of the IGFR extracellular (EC) sequence in the gag-IGFR fusion protein encoded by the retrovirus UIGFR have a modulatory effect on the biological and biochemical properties of the protein, four mutants, NM1, NM2, NM3, and NM4 of the EC sequence were constructed. NM1 lacks the entire 36 aa residues; NM2 lacks the N-terminal 16 aa residues (aa 870 to 885), including two potential N-linked glycosylation sites of the EC sequence; NM3 contains a deletion of the C-terminal 20 aa residues (aa 886 to 905) of the EC sequence; and NM4 contains N-to-Q substitutions at both N-linked glycosylation sites. NM1 was the strongest of the four mutants in promoting anchorage-independent growth of transfected chicken embryo fibroblasts, while NM2 and NM4 had weaker transforming potential than did the original UIGFR virus. Only NM1 and NM3 were able to induce sarcomas in chickens. The four NM mutant-transformed cells expressed the expected proteins with comparable steady-state levels. The in vitro tyrosine kinase activity of P53NM1 was about fourfold higher than that of the parental P57-75UIGFR, whereas NM2 and NM4 proteins exhibited four- to fivefold-lower kinase activities. Despite lacking the IGFR EC sequence, P53NM1 formed covalent dimers similar to those formed by the parental P57-75UIGFR. Increased phosphatidylinositol (PI) 3-kinase activity was found to be associated with the mutant IGFR proteins. Among NM4 proteins. Elevated tyrosine phosphorylation of cellular proteins of 35, 120, 140, 160, and 170 kDa was detected in all mutant IGFR-transformed cells. We conclude that the EC 36-aa sequence of IGFR in the gag-IGFR fusion protein exerts intricate modulatory effects on the protein's transforming and tumorigenic potential. The 20 aa residues immediately upstream of the transmembrane domain have an inhibitory effect on the tumorigenic potential of gag-IGFR, whereas N-linked glycosylation within the EC sequence appears to have a positive effect on the transforming potential of UIGFR. Increased in vitro kinase activity and, to a lesser extent, in vivo tyrosine phosphorylation as well as the elevated association of PI 3-kinase activity with IGFR proteins seem to be correlated with the transforming potential of IGFR mutant proteins.

Animals↗
PubMed1992

The insulinomimetic agents H2O2 and vanadate stimulate tyrosine phosphorylation of potential target proteins for the insulin receptor kinase in intact cells.

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate or H2O2 alone. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO.T cells) several proteins (e.g. pp180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mM-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 microM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO.YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.

Animals↗
PubMed1992

Pur-1, a zinc-finger protein that binds to purine-rich sequences, transactivates an insulin promoter in heterologous cells.

Purine-rich stretches of nucleotides (GAGA boxes) are often found just upstream of transcription start sites in many genes, including insulin. Mutational analysis suggests that the GAGA box plays an important role in transcription of the rat insulin I gene. We identify here at least four different proteins that bind specifically to the insulin GAGA box. Using a GAGA oligonucleotide, we have isolated a cDNA encoding a sequence-specific protein from a HIT (hamster insulinoma cell line) lambda gt11 library. This protein, which we designate Pur-1 (for purine binding), binds to the GAGA boxes of the rat insulin I and II genes and the human islet amyloid polypeptide gene. Pur-1 is a potent transactivator in both pancreatic and nonpancreatic cells. Furthermore, Pur-1 is able to activate an intact insulin promoter in HeLa cells, where it is normally inactive.

Amino Acid Sequence↗
PubMed1992

Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos.

A facile method for isolating genes that encode interacting proteins has been developed with a polypeptide probe that contains an amino-terminal extension with recognition sites for a monoclonal antibody, a specific endopeptidase, and a site-specific protein kinase. This probe, containing the basic region-leucine zipper dimerization motif of c-Fos, was used to screen a complementary DNA library. A complementary DNA that encoded a member of the basic-helix-loop-helix-zipper (bHLH-Zip) family of proteins was isolated. The complementary DNA-encoded polypeptide FIP (Fos interacting protein) bound to oligonucleotide probes that contained DNA binding motifs for other HLH proteins. When cotransfected with c-Fos, FIP stimulated transcription of an AP-1-responsive promoter.

Amino Acid Sequence↗
PubMed1992

Converting trypsin to chymotrypsin: the role of surface loops.

Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.

Acylation↗
PubMed1992

Transition-state characterization: a new approach combining inhibitor analogues and variation in enzyme structure.

A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.

Animals↗
PubMed1992

Onset of cell-specific gene expression in the developing mouse pancreas.

A central question in developmental biology has been the initiation of cell-specific gene expression and its temporal relationship to morphogenesis. We have coupled embryo microdissection with the exquisite sensitivity of the polymerase chain reaction to define the onset of cell-specific gene expression during pancreatic organogenesis. Using the precise assignment of gestational age by the number of somites in each embryo, we determined the onset of transcription of major genes of the endocrine and exocrine pancreas during mouse development to within 2-3 hr. Somatostatin mRNA was detected at the 10-somite stage throughout the foregut, consistent with the presence of somatostatin-producing cells throughout the adult gut. Mature mRNA for insulin and glucagon first appears surprisingly early, at the 20-somite stage in the wall of the embryonic foregut and is restricted to only the area of the duodenum from which the pancreas will arise 10-12 hr later. In contrast, exocrine gene transcription begins 24 hr after formation of the pancreatic diverticulum. Thus cell-specific gene expression in the endocrine pancreas begins in a "pre-morphogenetic phase." This early expression of insulin and glucagon could reflect the initiation of an endocrine cell lineage.

Amylases↗
PubMed1992

Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation.

The role of insulin-induced receptor autophosphorylation in its internalization was analyzed by comparing 125I-labeled insulin (125I-insulin) internalization in Chinese hamster ovary (CHO) cell lines transfected with normal (CHO.T) or mutated insulin receptors. In four cell lines with a defect of insulin-induced autophosphorylation, 125I-insulin internalization was impaired. By contrast, in CHO.T cells and in two other CHO cell lines with amino acid deletions or insertions that do not perturb autophosphorylation, 125I-insulin internalization was not affected. A morphological analysis showed that the inhibition is linked to the ligand-specific surface redistribution in which the insulin-receptor complexes leave microvilli and concentrate on nonvillous segments of the membrane where endocytosis occurs.

Animals↗