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Biomedical subjects

P M Kirwin

Publications and source records attributed to P M Kirwin.

10 recordsLinked to original sources
PubMed1992

Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.

Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine-28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.

3T3 Cells↗
PubMed1992

Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

Mutagenesis, Site-Directed↗
PubMed1989

Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex.

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.

Chlorophyll↗
PubMed1988

Transport of proteins into chloroplasts. Organization, orientation, and lateral distribution of the plastocyanin processing peptidase in the thylakoid network.

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen of the chloroplast. Maturation of preplastocyanin involves successive cleavages by a stromal peptidase and a distinct thylakoidal peptidase. In this report we have analyzed the precise location and orientation of the thylakoidal peptidase with respect to the thylakoid membrane. Experiments involving differential centrifugation of thylakoid extracts and sonication of isolated vesicles indicate that the peptidase is tightly bound to the thylakoid membrane but not intimately associated with any of the major thylakoid protein complexes. Analysis of the lateral distribution of the peptidase has shown that the enzyme is exclusively located in the non-appressed lamellae of the thylakoid network. The active site of the peptidase is on the lumenal face of the thylakoid membrane.

Chloroplasts↗
PubMed1987

Transport of proteins into chloroplasts. Partial purification of a thylakoidal processing peptidase involved in plastocyanin biogenesis.

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported across three membranes into the chloroplast thylakoid lumen. Processing to the mature size involves successive cleavages by a stromal and a thylakoidal peptidase. In this report we describe the partial purification and characterization of the thylakoidal peptidase involved. The enzyme has been purified 36-fold from Pisum sativum thylakoids after solubilization using Triton X-100. The peptidase processes the plastocyanin import intermediate to the mature size, but no further, and is capable of processing pre-plastocyanin to the mature size but at a lower rate. No detectable activity is displayed against non-chloroplast proteins or precursors of stromal proteins. The enzyme has a pH optimum of 6.5-7 and is activated by chelating agents such as EDTA and EGTA. No inhibitors of the peptidase have been found to date.

Biological Transport, Active↗
PubMed1977

The effect of descriptive anger expression, insult, and no feedback on interpersonal aggression, hostility, and empathy motivation.

In a test of the hypothesis that descriptive anger expression elicits less subsequent aggression and greater empathy than does aggressive insult, 60 male undergraduates were instructed to set varying levels of shock for an opponent during a series of competitive trials before and after hearing one of four types of taped comment. Results indicated that descriptive anger expression led to a significant decrease in aggression, while no feedback (opponent said nothing) led to an increase in aggression. Insult and no anger feedback resulted in little change in aggressive behavior. In contrast, descriptive anger expression, insult, and no anger feedback produced more residual hostility than did no feedback. Descriptive anger expression Ss appeared to be more motivated by empathy in setting shocks, as compared to the other three groups. Empathy was found to be generally associated with lower shock settings initially and with reductions in shock settings following the opponent's comments. Empathy, however, was not related to Ss' hostility ratings. The results tend to support the clinical utility of descriptive anger expression in improving interpersonal relations.

Aggression↗