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T Selwood

Publications and source records attributed to T Selwood.

17 recordsLinked to original sources
PubMed1998

Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

Benzamidines↗
PubMed1998

Recombinant expression of human mast cell proteases chymase and tryptase.

Expression of recombinant human chymase and tryptase was achieved in a baculovirus-insect cell system using a fusion protein construct. Recombinant baculovirus was produced with DNA coding for a NH2-ubiquitin-chymase-COOH or NH2-ubiquitin-tryptase-COOH fusion protein inserted immediately downstream of the signal sequence for the secreted envelope protein, glycoprotein 67. In each construct, the natural prepropeptide sequence of the protease was replaced by the amino acid sequence for the enterokinase cleavage site of trypsinogen. High Five insect cells infected with either of the modified baculovirus produced mg quantities of each fusion protein per liter of culture. Treatment of the chymase-fusion protein with enterokinase or the tryptase-fusion protein with enterokinase in the presence of a highly charged polysaccharide (dextran sulfate or heparin) produced enzymatically active proteases with properties of the native enzymes. A procedure for the purification of mg quantities of recombinant chymase from infected-cell medium is presented.

Animals↗
PubMed1997

Diverse effects of pH on the inhibition of human chymase by serpins.

Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.

Catalysis↗
PubMed1995

Structural changes associated with the spontaneous inactivation of the serine proteinase human tryptase.

Human skin tryptase, a serine proteinase stored within mast cell secretory granules, rapidly loses enzymatic activity in solutions of physiological salt concentration, pH, and temperature. The inactivation of tryptase can be slowed and even reversed by addition of heparin, a highly sulfated glycosaminoglycan also found in the secretory granules. These properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underlying the functional instability of tryptase, circular dichroism (CD) and analytical ultracentrifugation were used to investigate structural changes during spontaneous inactivation. The CD spectra of active and spontaneously inactivated tryptase are different, particularly in the region around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotrypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activity resulting from spontaneous inactivation was accompanied by a diminution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activity loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of decay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase. The return of the CD signal appeared to be more rapid than the return of enzymatic activity, thereby suggesting the presence of an unidentified step which is rate-limiting for activity return (and loss) and subsequent (prior) to the CD change accompanying activity loss. Ultracentrifugation analysis of tryptase showed a marked change in its association state upon inactivation. Sedimentation equilibrium under stabilizing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mixture of species was evident, which was characterized as monomers and tetramers in equilibrium. These results demonstrate that spontaneous inactivation of tryptase is associated with reversible conformational changes and that a consequence of inactivation is the formation of a destabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tryptase and chymotrypsin.

Chymases↗
PubMed1994

Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin.

The physiological alpha-oxoaldehyde methylglyoxal binds and modifies arginine, lysine, and cysteine residues in proteins. The kinetics and mechanism of these reactions were investigated with N alpha-acetylamino acids and bovine serum albumin at pH 7.4 and 37 degrees C. The reaction of methylglyoxal with N alpha-acetylarginine involved the initial reversible formation of glycosylamine and 4,5-dihydroxy-5-methylimidazolidine derivatives, with further slow irreversible conversion to an imidazolone, N alpha-acetyl-N delta- (5-methyl-4-imidazolon-2-yl)ornithine. The imidazolone was fluorescent with an excitation lambda max value of 320 nm and an emission lambda max value of 398 nm. Methylglyoxal reacted reversibly with N alpha-acetyllysine to form glycosylamine and bisglycosylamine derivatives. Further reaction of these glycosylamines occurred to form brown, fluorescent oligomers that were not characterized. Methylglyoxal reacted rapidly and reversibly with N alpha-acetylcysteine to form the hemithioacetal adduct. The reaction of methylglyoxal with bovine serum albumin (BSA) at pH 7.4 and 37 degrees C involved the reversible and irreversible formation of methylglyoxal-BSA adducts. Irreversible modification of BSA occurred mainly on arginine residues to form imidazolone. The formation of methylglyoxal-modified proteins involves glycoxidation leading to advanced glycation end product-like fluorescence. It is expected to be increased in diabetes mellitus and may be linked to the development of diabetic complications.

Acetylcysteine↗
PubMed1994

The reaction of methylglyoxal with aminoguanidine under physiological conditions and prevention of methylglyoxal binding to plasma proteins.

Increased formation of methylglyoxal in clinical diabetes mellitus and metabolism by the glyoxalase system has been linked to the development of clinical complications of diabetes: retinopathy, neuropathy and nephropathy. Aminoguanidine has been proposed as a prophylactic agent for preventive therapy of diabetic complications. Methylglyoxal reacted with aminoguanidine under physiological conditions to form two isomeric triazines, 3-amino-5-methyl-1,2,4-triazine and 3-amino-6-methyl-1,2,4-triazine. The mean second order rate constant for the reaction of methylglyoxal with aminoguanidine, kMG.AG = 0.39 +/- 0.06 M-1 sec-1 at pH 7.4 and 37 degrees. Under these conditions, no methylglyoxal bisguanylhydrazone was detected. Aminoguanidine prevented the irreversible modification of human plasma protein by a physiological concentration of methylglyoxal (1 microM); the median inhibitory concentration IC50 value of aminoguanidine was 203 +/- 16 microM (N = 28). The scavenging of methylglyoxal by aminoguanidine may contribute to the beneficial effects of aminoguanidine in the prevention of vascular pathogenesis in diabetes.

Blood Proteins↗
PubMed1990

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗
PubMed1988

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.

Arabinose↗
PubMed1988

The relationship of basal cell carcinomas and squamous cell carcinomas to solar keratoses.

Six thousand four hundred sixteen people aged 40 years and over from three different locations in Victoria (Australia) were examined on the hands, forearms, head, and neck for the presence of solar keratoses and basal (BCCs) and squamous cell carcinomas (SCCs). Analysis of the relationship between these tumors revealed that the factors which predicted the likelihood of developing a solar keratosis were essentially the same as those that predicted the likelihood of developing a BCC and/or an SCC. These were age, sex, years of residence in Australia, indoor or outdoor occupation, tanning ability, propensity to sunburn, and location of residence. The presence of a coexisting solar keratosis was necessary for the development of an SCC in contrast to the development of a BCC. The findings suggest that unlike BCCs, the majority of SCCs in light-exposed areas may arise from preexisting solar keratoses. Whereas the prevalence of BCCs and SCCs was relatively constant in the three locations, the prevalence of solar keratoses differed markedly in direct relation to the degree of isolation. This suggests that solar keratoses are a more sensitive indicator of sunlight exposure than invasive carcinoma.

Adult↗
PubMed1980

Sterilization operations in Australia.

The proportion of males and females in Australia having privately performed sterilization operations has been estimated from 1976 operation rates for each age group and for each State. These figures indicate that 60% of Australian couples (maximum: 74% in Western Australia; minimum: 53% in Victoria) will choose this form of birth control during their fertile lifetime. A general practitioner survey in March, 1978, indicated this proportion would be an underestimate. If this rate of sterilization continues, we will challenge the United States as the country with the highest prevalence of sterilized couples.

Adult↗
PubMed1980

Artificial donor insemination frozen versus fresh semen.

The results of artificial insemination by fresh and frozen donor semen (A.I.D.) are reported. Life table conception results were 66.3 +/- 3.8 after 6 months for the fresh semen group. Similar results for 2 consecutive programmes of A.I.D. of frozen semen were 44.0 +/- 4.6 and 64.2 +/- 3.4, respectively. The latter group's cumulative pregnancy rate is high and probably reflects improvement in both sperm preservation and insemination techniques. Using improved freezing techniques, the results of A.I.D. with frozen semen are equal to those with fresh semen.

Actuarial Analysis↗
PubMed1978

The tortuous tube: pregnancy rate following laparoscopy and hydrotubation.

256 infertile women whose ovulatory and related male fertility factors were normal were investigated by diagnostic laparoscopy and dye hydrotubation. The tubes were objectively classed into 4 groups--normal, patent with macroscopic tubal adhesions, patent with one tortuosity, and patent with multiple tortuosities. The latter group had a highly significant response to the above procedures and 66% were pregnant within 1 year and 81% within 2 years. No significant effect was found in the other 3 groups. This multiple tortuosity group represents the type of tube that significantly responds to treatment inherent in all tubal patency tests. Its pathogenesis and management are discussed.

Adnexal Diseases↗