PubMed Health⌕ Search

Biomedical subjects

W J Rutter

Publications and source records attributed to W J Rutter.

At least 55 records · Page 3Linked to original sources
PubMed1992

Guanidine derivatives restore activity to carboxypeptidase lacking arginine-127.

Arg-127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A-catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg-127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 10(4) for R127A). Therefore, Arg-127 stabilizes the enzyme-transition state complex but not the ground state enzyme-substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692-20698). The addition of guandine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz-gly(o)phe by 10(2) without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz-phe-alaP(o)ala is increased similarly by 10(2); in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg-127 in stabilizing the rate-limiting transition state. Hydrolysis of Bz-gly-(o)phe by wild-type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg-127 specifically and in a productive conformation for catalysis.

Animals↗
PubMed1992

Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex.

The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. We have isolated several cDNAs encoding proteins that bind to the FLAT element. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich "LIM" domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. We conclude that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional minienhancer complex.

Amino Acid Sequence↗
PubMed1992

Human skin mast cell carboxypeptidase: functional characterization, cDNA cloning, and genealogy.

We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did bovine pancreatic carboxypeptidase A (CPA) against carboxyl-terminal leucine residues, about equal activity with phenylalanine and tyrosine residues, and no activity with tryptophan or alanine. To correlate kinetic data with structure, we isolated and sequenced a cDNA encoding MC-CPA from human skin, and directly sequenced 30% of the purified protein. These sequences agreed with that of human lung MC-CPA, and further support the evidence for a single MC-CPA gene in humans. Four amino acid replacements, resulting in a net positive change in non-hydrogen atoms in the S1' subsite of MC-CPA, were associated with less alteration in substrate specificity, relative to bovine CPA, than might be expected from studies using rat CPA1 and CPA2. We noted two consensus N-linked glycosylation sites in human MC-CPA that are not found in rat and mouse MC-CPA, or in bovine CPA; that at least one of these sites is glycosylated in vivo was verified by N-glycosidase F treatment, lentil lectin binding, and Concanavalin A-Sepharose chromatography. Evolutionary trees constructed from the known carboxypeptidase sequences suggested that MC-CPA most likely evolved from a carboxypeptidase B-like enzyme, independent of the pancreatic CPA. Thus, in the carboxypeptidase gene family, MC-CPA displays a unique genealogy and several amino acid replacements in its S1' binding pocket that result in substrate specificity quite similar to bovine CPA.

Amino Acid Sequence↗
PubMed1992

Enhancement of transforming potential of human insulinlike growth factor 1 receptor by N-terminal truncation and fusion to avian sarcoma virus UR2 gag sequence.

The human insulinlike growth factor 1 (hIGF-1) receptor (hIGFR) is a transmembrane protein tyrosine kinase (PTK) molecule which shares high sequence homology in the PTK domain with the insulin receptor and, to a lesser degree, the ros transforming protein of avian sarcoma virus UR2. To assess the transforming potential of hIGFR, we introduced the intact and altered hIGFR into chicken embryo fibroblasts (CEF). The full-length hIGFR cDNA (fIGFR) was cloned into a UR2 retroviral vector, replacing the original oncogene v-ros. fIGFR was able to promote the growth of CEF in soft agar and cause morphological alteration in the absence of added hIGF-1 to medium containing 11% calf and 1% chicken serum. The transforming ability of hIGFR was not further increased in the presence of 10 nM exogenous hIGF-1. The 180-kDa protein precursor of hIGFR was synthesized and processed into alpha and beta subunits. The overexpressed hIGFR in CEF bound hIGF-1 with high affinity (Kd = 5.4 x 10(-9) M) and responded to ligand stimulation with increased tyrosine autophosphorylation. The cDNA sequence coding for part of the beta subunit of hIGFR, including 36 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains, was fused to the 5' portion of the gag gene in the UR2 vector to form an avian retrovirus. The resulting virus, named UIGFR, was able to induce morphological transformation and promote colony formation of CEF with a stronger potency than did fIGFR. The UIGFR genome encodes a membrane-associated, glycosylated gag-IGFR fusion protein. The specific tyrosine phosphorylation of the mature form of the fusion protein, P75, is sixfold higher in vitro and threefold higher in vivo than that of the native IGFR beta subunit, P95. In conclusion, overexpression of the native or an altered hIGFR can induce transformation of CEF with the gag-IGFR fusion protein possessing enhanced transforming potential, which is consistent with its increased in vitro and in vivo tyrosine phosphorylation.

Animals↗
PubMed1992

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators.

Amyloid↗
PubMed1992

Tissue and species differences in bile salt-dependent neutral cholesteryl ester hydrolase activity and gene expression.

Enzymatic activity and mRNA abundance for neutral bile salt-dependent cholesteryl ester hydrolase (CEH) were determined in rat and rabbit tissues. In rat liver and intestine, enzyme activity and mRNA levels varied independently. Particularly striking in most tissue samples was the absence of detectable CEH mRNA in the presence of enzymatic activity, suggesting that there was an exogenous source of enzyme. Rabbits differed from rats in four ways. First, neither CEH activity nor mRNA was present in any liver sample. Second, CEH mRNA was present in nearly all intestinal samples, and its abundance tended to correlate with enzymatic activity. Third, rabbit CEH mRNA was approximately 250 bases shorter than the rat message. Fourth, we have previously shown that rat plasma contains CEH activity, whereas in the present studies, rabbit plasma did not contain such activity. Overall, our studies indicate that CEH activity in rat liver, intestine, and plasma can be derived exogenously, most likely from the uptake and transport of pancreatic enzyme. In contrast, in rabbit the lack of CEH activity in plasma and liver and the capacity of the intestine for in situ synthesis of CEH suggest that this animal does not have the same ability to distribute pancreatic CEH. These species differences in CEH metabolism may partly explain the greater susceptibility of rabbit tissues to accumulate cholesteryl esters.

Animals↗
PubMed1991

Structural evolution of an enzyme specificity. The structure of rat carboxypeptidase A2 at 1.9-A resolution.

The structure of rat carboxypeptidase A2 (CPA2), which has a unique specificity for tryptophan-containing COOH-terminal peptides, has been determined in an unliganded state at 1.9-A resolution and refined to a crystallographic R-factor of 18.3%. Comparison of the structure of CPA2 with that of bovine carboxypeptidase A (referred to here as CPA1) reveals that the specificity of the former for larger amino acids probably arises from two amino acid replacements within the binding cavity (Thr268----Ala and Leu203----Met), coupled with differences in the positions of conserved residues in a surface loop on one face of the specificity pocket. The position of the reactive-site surface loop may be affected also by a disulfide bridge between Cys210 and Cys244. In this unliganded form of the enzyme, Tyr248 takes up a position interior to the specificity pocket and is distinct from that observed in bovine CPA1. The structural differences between CPA1 and CPA2 correlate strongly with crystallographically determined temperature factors and thus appear to be largest where the enzyme is flexible.

Amino Acid Sequence↗
PubMed1991

A pancreatic exocrine cell factor and AP4 bind overlapping sites in the amylase 2A enhancer.

A factor found in pancreatic exocrine cell lines and pancreatic nuclei binds selectively to the alpha-amylase 2A transcriptional enhancer. Pancreatic exocrine cell extracts protect asymmetrically an unusually large, 35 base pair region from DNase I digestion in vitro, suggesting the involvement of a multimeric DNA binding complex. We show that this region of the enhancer contains a major affinity recognition sequence for the HeLa transcription factor AP4. A 4 base pair mutation in the enhancer sequence shown previously to abolish activity in vivo [Boulet, A. M., Erwin, C. R., & Rutter, W. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3599-3603] abolishes AP4 binding in vitro and weakens but does not eliminate the binding of adjacent enhancer factors. Further, sequences similar to the AP4 binding site are found within a consensus sequence of most pancreatic exocrine genes (Boulet et al., 1986). We have identified three AP4 binding sites in the pancreatic elastase gene: one occurs in the consensus sequence of the enhancer. Thus, protein(s) with the binding selectivity of AP4 may play a role in the expression of the pancreatic exocrine gene family.

Amylases↗
PubMed1991

Molecular basis of the activation of the tumorigenic potential of Gag-insulin receptor chimeras.

A previous study showed that the human insulin receptor (IR) could be activated by insertion of a 3' portion of the cDNA encoding the beta subunit into a retrovirus genome to form a Gag-IR fusion protein. While capable of transforming cells in culture, this IR cDNA-containing virus, called UIR, was not able to induce tumors in animals. Subsequently, we isolated a spontaneous sarcomagenic variant called UIR19t from the parental UIR. UIR19t was molecularly cloned, sequenced, and found to harbor two mutations. A 44-amino acid deletion immediately upstream from the transmembrane domain of the Gag-IR fusion protein removes all the extracellular sequence of the IR remaining in the original UIR construct. In addition, a single nucleotide deletion at the 3' end results in truncation and replacement of the carboxyl-terminal 12 amino acids by 4 new amino acids. The specific kinase activity of UIR19t is 4- to 5-fold higher than that of the parental UIR. However, no new cellular substrates were detected in UIR19t-transformed cells as compared to UIR cells. Viruses containing either the 5' or the 3' deletion mutation were constructed and assessed for their biological function. Our data indicate that the 5' deletion alone is sufficient to confer tumorigenic ability. We conclude that sequence immediately upstream from the transmembrane domain imposes a negative effect on the transforming and tumorigenic potential of the Gag-IR fusion protein.

Amino Acid Sequence↗
PubMed1991

Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes.

Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes.

Amylases↗
PubMed1991

Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer.

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.

Amino Acid Sequence↗
PubMed1990

Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures.

To study the regulation of insulin gene expression by physiological regulators, primary cultures of rat islet cells were transfected with portions of the rat insulin I gene 5'-flanking sequence linked to the reporter gene chloramphenicol acetyltransferase (CAT). Incubation of the cells in increasing glucose concentrations led to a parallel increase in both CAT activity and CAT mRNA levels. Pretreatment of the cells with the beta-cell-specific toxin streptozotocin reduced CAT activity 97%. Beta-Cell-specific expression of CAT was also demonstrated by co-staining the transfected cells with antisera to both CAT and insulin. Experiments showing a reduction in the response to glucose in the presence of the calcium channel blocker verapamil suggest that calcium plays a role in the glucose response, possibly via regulation of factors interacting with this limited portion of the insulin gene.

Animals↗
PubMed1990

Arginine 127 stabilizes the transition state in carboxypeptidase.

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.

Animals↗
PubMed1990

The cysteine-rich domains of the insulin and insulin-like growth factor I receptors are primary determinants of hormone binding specificity. Evidence from receptor chimeras.

To delineate the structural determinants of the insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) which affect hormone binding specificity we have constructed seven chimeric receptor cDNAs and stably expressed them in Chinese hamster ovary cells. Clonal cell lines expressing high levels of each receptor chimera were analyzed for insulin and insulin-like growth factor I (IGFI) binding activity. Measurements of hormone binding and immunoprecipitation of metabolically labeled receptors showed that all chimeras were properly processed and expressed at the cell surface. The binding data indicate that 56 amino acids of the IR and 52 amino acids of the IGFIR located in corresponding regions of the cysteine-rich domains are the primary determinants of hormone binding specificity. These regions are located between amino acids Asn-230 and Ile-285 on the IR and between His-223 and Met-274 on the IGFIR. In addition, the alpha IR-3 antibody, which competes for IGFI binding, was found to interact with the same 52 amino acids of the IGFIR which determines hormone specificity. Other antibodies which interfere with insulin binding (5D9, MC51, and MA20) interact with epitopes in the COOH-terminal 288 amino acids of the alpha-subunit. We conclude that 56 and 52 amino acids of the cysteine-rich domains of the IR and IGFIR contain the major determinants of hormone binding specificity although other more COOH-terminal regions of both receptors contribute to hormone binding.

Amino Acid Sequence↗