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M A Phillips

Publications and source records attributed to M A Phillips.

At least 55 records · Page 3Linked to original sources
PubMed1995

Modeling of the spatial structure of eukaryotic ornithine decarboxylases.

We used sequence and structural comparisons to determine the fold for eukaryotic ornithine decarboxylase, which we found is related to alanine racemase. These enzymes have no detectable sequence identity with any protein of known structure, including three pyridoxal phosphate-utilizing enzymes. Our studies suggest that the N-terminal domain of ornithine decarboxylase folds into a beta/alpha-barrel. Through the analysis of known barrel structures we developed a topographic model of the pyridoxal phosphate-binding domain of ornithine decarboxylase, which predicts that the Schiff base lysine and a conserved glycine-rich sequence both map to the C-termini of the beta-strands. Other residues in this domain that are likely to have essential roles in catalysis, substrate, and cofactor binding were also identified, suggesting that this model will be a suitable guide to mutagenic analysis of the enzyme mechanism.

Amino Acid Sequence↗
PubMed1994

Formation of functional cross-species heterodimers of ornithine decarboxylase.

The two active sites in ornithine decarboxylase (ODC) are formed at the dimer interface with Lys-69 and Cys-360 contributing to each active site from opposite monomers [Tobias, K. E., & Kahana, C. (1993) Biochemistry 32, 5842-5847]. To gain insight into the organization of the substrate binding site and the nature of the dimer interface, analysis of ornithine decarboxylase from two parasitic protozoa, Trypanosoma brucei and Leishmania donovani, and from mouse was undertaken. Though T. brucei and mouse ornithine decarboxylase share only 60% sequence identity, the cross-species heterodimers form spontaneously, as measured by the restoration of enzyme activity upon mixing inactive K69A and C360A mutant enzymes. Thus, the amino acid composition of the dimer interface is apparently highly conserved between the T. brucei and mouse enzymes. Cross-species heterodimers were not formed between either T. brucei or mouse ODC and L. donovani ODC. Unlike the mouse and T. brucei ODC, the subunits of L. donovani ODC are not in rapid equilibrium, and incubation with a denaturant is required to induce reassociation. Kinetic analysis of the wild-type mouse and parasite ODCs revealed differences in the substrate binding sites between the three enzymes. The substrate binding properties of the restored active site in the T. brucei:mouse cross-species heterodimer mimic the characteristics of the wild-type enzyme from the species which contributes the subunit with a functional Lys-69.

Amino Acid Sequence↗
PubMed1994

Cyclic peptides as proteases: a reevaluation.

A recent report [Atassi, M. Z. and Manshouri, T. (1993) Proc. Natl. Acad. Sci. USA 90, 8282-8286] described the design and synthesis of two 29-amino acid cyclic peptides that were reported to hydrolyze both ester and amide bonds with chymotrypsin-like or trypsin-like specificity. We have synthesized the trypsin-mimic peptide (TrPepz) and detect no activity toward either ester or peptide substrates. The same result was independently obtained by Wells et al. [Wells, J. A., Fairbrother, W. J., Otlewski, J., Laskowski, M., Jr., & Burnier, J. (1994) Proc. Natl. Acad. Sci. USA 91, 4110-4114.] Additionally, we found that Atassi and Manshouri failed to obtain accurate kinetic constants for trypsin- and chymotrypsin-catalyzed ester hydrolysis because the high concentrations of trypsin and chymotrypsin that they report using would have prevented evaluation of initial rates. These findings are incompatible with the claims, reported by Atassi and Manshouri, that TrPepz has trypsin-like activity.

Amino Acid Sequence↗
PubMed1994

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences.

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.

Biological Evolution↗
PubMed1994

Impact of psychological and physical health, stressful events, and social support on subsequent mental health help seeking among older adults.

Factors that influence mental health help seeking among adults 55+ years were examined prospectively. A discriminant analysis significantly differentiated between 120 older adults needing and seeking services and a comparison group of 120 older adults not needing services. Prior to having sought help, help seekers demonstrated poorer psychological well-being, reported more physical health problems, reported a higher level of unpleasant stressful events, and perceived greater deficits in the amount of social support available to them in time of need. The vast majority of these older help seekers sought help for their mental health problems from a medical doctor rather than from a mental health center or clinic or from a minister. Significantly more help seekers than nonseekers experienced stressful events involving bereavement, social and economic loss, and new physical illness.

Activities of Daily Living↗
PubMed1994

Decreased systemic thromboxane A2 biosynthesis in normal human subjects fed a salmon-rich diet.

Nine normal male subjects were fed a reference diet typical of that consumed in the United States and a diet containing approximately 450 g salmon (salmon-rich diet). The salmon diet contained approximately 6 g omega 3 fatty acids that comprised 2.0% energy intake/d for each individual. The percent energy contribution of protein, carbohydrate, and fat (19%, 56%, and 25%, respectively) was identical for the two diets. Urinary excretion of 2,3-dinor-thromboxane B2 was significantly lower (0.74 +/- 0.26 ng/24 h) with the salmon diet compared with the reference diet (0.95 +/- 0.31 ng/24 h). In addition, in vitro generation of thromboxane B2 in response to collagen-stimulated aggregation of platelet-rich plasma from subjects consuming the salmon diet was reduced (1.87 +/- 0.79 ng/mL) compared with subjects consuming the reference diet (3.10 +/- 1.81 ng/mL). Urinary 2,3-dinor-6-oxo-prostaglandin F1 alpha excretion in subjects was not significantly different between the salmon diet (0.69 +/- 0.33 ng/24 h) and the reference diet (0.81 +/- 0.44 ng/24 h).

Adult↗
PubMed1994

Arsenate perturbation of human keratinocyte differentiation.

Treatment of cultured malignant human keratinocytes with sodium arsenate greatly suppressed expression of involucrin, a specific marker of keratinocyte differentiation. This action was primarily attributable to inhibition of involucrin transcription according to message run-on and stability measurements. Involucrin was suppressed in nontumorigenic keratinocytes as well, although the efficacy of suppression was less dramatic in cells derived from clinically normal epidermis. Several transition metal oxyanions (vanadate, molybdate, and tungstate) also substantially suppressed involucrin expression, but okadaic acid was ineffective. Immunoblotting detected marked increases in tyrosine phosphorylation of several proteins as a consequence of arsenate treatment of the cultures, while mobility shift analysis revealed a dramatic loss of DNA binding by the transcription factor AP2. These findings support a proposed role for altered levels of protein tyrosine phosphorylation in keratinocyte differentiation. They also suggest that arsenate perturbs the differentiation program in target cells by altering this phosphorylation level and transcription factor activity.

Arsenates↗
PubMed1993

Keratinocyte transglutaminase membrane anchorage: analysis of site-directed mutants.

Keratinocyte transglutaminase is anchored on the cytosolic side of the plasma membrane by fatty acid thioesterification near the amino terminus, a process which is seen to occur within 30 min of synthesis. The importance of a cluster of five cysteines (residues 47, 48, 50, 51, and 53) where acylation was presumed to occur is now demonstrated by site-directed mutagenesis. Transglutaminase mutants in which the cluster is deleted or the cysteines are all converted to alanine or serine are cytosolic. Partial replacement of the cluster, leaving two contiguous cysteines, is sufficient to confer membrane anchorage, while a single cysteine is only partially effective. As demonstrated with a soluble transglutaminase mutant, membrane anchorage confers susceptibility of the amino-terminal region to phorbol ester-stimulated phosphorylation. Attachment of 105 residues from the transglutaminase amino terminus to involucrin, a highly soluble protein, results in membrane anchorage of the hybrid protein. Attachment of the cysteine cluster alone does not result in membrane attachment of involucrin, but a 32-residue segment containing this cluster is sufficient. Stable transfectants of the human transglutaminase in mouse 3T3 cells are membrane-bound, indicating the fatty acid transacylation is not keratinocyte-specific.

3T3 Cells↗
PubMed1993

Dietary modification of omega 6 fatty acid intake and its effect on urinary eicosanoid excretion.

A group of women were fed two separate diets in a crossover study and urinary eicosanoids were quantified. One diet contained 3.1% of total energy (en%) as polyunsaturated fatty acids (3.0 en% linoleic acid) and the other contained 8.4 en% polyunsaturated fatty acids (8.3 en% linoleic acid). Carbohydrate replaced fat in the low-polyunsaturated-fat diet. No changes were observed in the urinary excretion of 6-oxo-prostaglandin F1 alpha, its 2,3-dinor metabolite or thromboxane B2 by subjects on either of the diets. Urinary 2,3-dinor-thromboxane B2 excretion was lower (206.5 ng/24 h) when subjects were fed the high-omega 6 polyunsaturated fatty acid diet when compared with the lower-omega 6 polyunsaturated fatty acid diet (275.3 ng/24 h). Conversely, urinary prostaglandin E2 was higher (139.2 ng/g creatinine) during the higher-omega 6 polyunsaturated fatty acid diet when compared with the lower-omega 6 polyunsaturated fatty acid diet (94.4 ng/g creatinine).

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Genomic structure of keratinocyte transglutaminase. Recruitment of new exon for modified function.

The gene for keratinocyte transglutaminase (TGK) spans 14 kilobase pairs and contains 15 exons. Many features of the TGK gene are very similar, if not identical, to those of the gene encoding the catalytic subunit of human clotting factor XIII: they have the same number of exons, corresponding introns always interrupt the coding region in the same phase of the codon, and most exons are of similar size (10 or 15 are exactly the same size). In these respects, the TGK and factor XIII catalytic subunit genes resemble each other more than either resembles the gene for erythrocyte band 4.2, a noncatalytic transglutaminase superfamily member. Exon II in both the TGK and factor XIII genes encodes an amino-terminal extension of nonhomologous sequence which in each protein confers a specialized function (membrane anchorage or activation of cross-linking, respectively). This suggests that the evolution of these genes included recruitment of a new exon to modify the enzyme action. Southern blots of genomic DNA reveal the presence of a TGK-like gene in birds, amphibians, and fish, but not in flies.

Amino Acid Sequence↗
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

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

The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters.

Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5'-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the pho A promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.

Amino Acid Sequence↗
PubMed1991

The operational costs of spraying residual insecticides: a case-study from Nepal.

Little attention has been paid to the cost implications of different insecticides for malaria control, particularly to the local (non-insecticide) costs. This study in Nepal explored the significance of operational costs in relation to insecticide cost and examined the extent to which operational costs varied for DDT, malathion and Ficam. A detailed analysis was done of costs relating to spraying, transport and safety. The study found that the operational cost differences were relatively small in comparison with the costs of the chemicals themselves and the cost of transporting them to Nepal, though DDT and Ficam had significantly lower operational costs than malathion. Operational costs were paid mainly by the Nepalese government and insecticide and external freight costs by donors.

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

Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation.

Ornithine decarboxylase (ODC) is a key enzyme in polyamine biosynthesis. Mouse ODC is rapidly degraded in mouse cells, whereas ODC within Trypanosoma brucei, a protozoan parasite infesting cattle, is stable. We have expressed cloned ODC genes of both T. brucei and mouse in ODC-deficient Chinese hamster ovary (CHO) cells. The T. brucei enzyme is stable, whereas the mouse ODC similarly expressed in CHO cells is unstable. This shows that the observed difference in intracellular stability is a property of the ODC protein itself, rather than the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome and the carboxyl terminus of mouse ODC is rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxyl terminus determine its stability.

Amino Acid Sequence↗
PubMed1990

Primary structure of keratinocyte transglutaminase.

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

Amino Acid Sequence↗