No description available.

Staphylococcus aureus protease V8 (SPV8), also known as Endoproteinase Glu-C (EC 3.4.21.19), is an enzyme isolated from the bacteria Staphylococcus aureus. This unusual enzyme has been found to cleave specifically at glutamyl and aspartyl peptide bonds and has been used as a tool in the preparation of protein substrates for amino acid sequence analysis. SPV8 has been reported to show some stability toward various denaturants (Drapeau, G.R. (1977) Methods in Enzymology_, 47:189-191). In order to more adequately assess the denaturant stability of SPV8, the effect of guanidine hydrochloride (HC1), a common protein denaturant, on the proteolytic action of SPV8 was studied. The extent of cleavage of the glutamyl peptide bond in adrenocorticotropic hormone 1-10 (ACTH 1-10) was found to decrease with increasing concentrations of guanidine HC1.At 22°C in the presence of 3.0 M guanidine HCl, only 25% of SPV8's proteolytic activity was retained. In the presence of 4.0, 5.0, or 6.0 M guanidine HC1, virtually all proteolytic activity toward the glutamyl bond of ACTH 1-10 was lost, presumably due to the inactivation of the protease by denaturation or increased autolysis mediated by the guanidine HC1. At temperatures above 22°C, SPV8 was more susceptible to inactivation by guanidine HC1. Thus SPV8 appears to retain some proteolytic activity in the presence of guanidine HC1, but only at concentrations less than 4.0 M. There was no difference in the proteolytic activity of SPV8 toward the glutamyl peptide bond of ACTH 1-10 when incubation was carried out in ammonium bicarbonate buffer (pH 7.80), phosphate buffer (pH 7.80), or Tris-HC1 buffer (pH 7.80). The presence of 1 mM calcium chloride in the 3.0 M guanidine HC1/phosphate buffer solution enhanced the enzymatic action of SPV8. The presence of 1 mM calcium chloride in Tris-HC1 buffer (pH 7.80) does not effect the proteolytic activity of SPV8 at 22°C. However, there was slight reduction in SPV8's enzymatic action toward ACTH 1-10 when the 1 mM calcium chloride was present in the 3.0 M guanidine HC1/ammonium bicarbonate buffer (pH 7.80) solution. / Department of Chemistry

Thesis (M.S.)--Rochester Institute of Technology, 1991. / Spine title: Active site configuration of GDH. Typescript. References: leaves 73-75.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 129-137).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Preliminary observations of the restricted regionalization of the enzyme 2' ,3'-cyclic nucleotide-3’ -phosphohydrolase led to an investigation of the subcellular regionalization of this enzyme in cerebral white matter. Since bovine corpus callosum contained eighteen times as much enzyme activity as grey matter, an association of the enzyme with myelin was suggested. Subsequent fractionation of bovine cerebral white matter by sucrose density gradient according to the procedure of Autilio, Norton, and Terry for the purification of myelin (1), showed that greater than 60% of the total activity was associated with the myelin rich fractions. In order to fractionate cerebral white matter more thoroughly, a modified De Robertis fractionation procedure was utilized allowing for separation of nuclear, mitochondrial, and microsomal pellets by differential centrffugation (2). Phosphohydrolase activity was distributed in all fractions, and electron microscopy demonstrated the presence of myelin in all of these fractions. Subsequent fractionation of these primary fractions on a discontinuous sucrose density gradient, showed essentially all of the phosphohydrolase activity in the lightest fraction at the top of each gradient. This band was comprised primarily of myelin figures as verified by electron microscopy. These studies indicated that the enzyme was associated with myelin. The foregoing result was further supported by a study of the increase in enzyme activity during myelination in rats. Myelination is known to occur early in the life of the rat, being initiated a few days after birth, entering a rapid phase of onset at about 10 days and being essentially complete after 50 days (3). Cholesterol was shown to increase in a corresponding manner indicating that myelination was indeed proceeding. Further evidence that the enzyme is associated with myelin came from an investigation of mutant mice. Quaking mice have been shown to be deficient in myelin, containing, according to Bauman and co-workers (4), only 62% of the normal galactolipid levels. Since galactolipids are presently accepted markers for myelin, and since adult quaking mice had 50% of the control enzyme activity, in agreement with the published galactolipid values, it was thought not unlikely that the two phenomena were related. This result also inferred an association of the enzyme with myelin. In attempt to further uncover the physiological role of the 2’,3'-cyclic nucleotide-3'-phosphqhydrolase, investigations have been directed towards elucidation of the substrate specificity of the enzyme. Uridine and guanosine-2’,3'-cyclic phosphothioates, kindly donated by Dr. Fritz Eckstein of the Max Planck Institut für Experimentelle Medizin, were hydrolyzed at rates of l.4 and l4.3% that of adenosine-2’,3'-cyclic phosphate. Cyclic inositol phosphate, synthesized from inositol-2-phosphate in the presence of dicyclohexylcarbodi-imide and pyridine, and glucose-l,2-cyclic phosphate, synthesized from (formula omitted)-D-glucose-l-phosphate, in a similar manner, were not hydrolyzed td any measureable extent. Preliminary results also show that ribose cyclic phosphates are not hydrolyzed, indicating a requirement for a purine or pyrimidine ring in the substrate molecule. These results are discussed with respect to their possible physiological significance. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Call number: LD2668 .T4 1984 S87 / Master of Science

The biosynthesis of Aflatoxin B1 (AFB1) has been the subject of conflicting speculation and numerous reviews. The currently accepted scheme for the aflatoxin pathway is based on data obtained from feeding studies using isotopically labelled precursors. In these studies the conversion of possible intermediate metabolites to AFBl by mutants of Aspergillus parasiticus illustrated their role as biogenetic precursors. Currently there is now agreement on the identity of most of the intermediate Illetabolites involved in the biosynthesis of AFB1. However, there is a lack of clarity on the details of AFB1 biosynthesis including the conversion of sterigmatocystin (ST) to AFB1 via the metabolite O-methylsterigmatocystin (OMST). There is no clear cut evidence of the metabolic role of OMST, i.e., either it is a compulsory intermediate or a shunt metabolite and hence part of a metabolic grid. In order to investigate this step in AFBl biosynthesis, ST was isolated from surface cultures of A. versicolor (M1101) and purified by silica gel column chromatography and repeated recrystallisation. Sterigmatocystin was characterised by thin layer chromatography (t.1.c.), low resolution mass spectrometry (M.S) and nuclear magnetic resonance spectroscopy (N.M.R). A series of seven derivatives of the free hydroxyl group of ST were synthesised by known chemical reactions, purified by silica gel column chromatography and characterised by high resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. A high pressure liquid chromatography (HPLC) method was developed using a fluorescence detector. The optimum parameters for the separation of the four major aflatoxins, namely AFBl, AFB2, AFGl and AFG2, using trifluoroacetic acid as the derivatising reagent, were obtained for a reversed phase Prodigy C18 column with a mobile phase of water: acetonitrile: isopropanol: acetic acid (8: 1: 0.5: 0.5, v/v). Feeding studies, using whole cells of A. parasiticus (WhI-11-105), showed that ST and the ST derivatives were converted to AFB1. A time courser study for the conversion of ST and selected ST derivatives to AFB1 indicated a decrease in the rate of conversion in the order: a-propyl sterigmatocystin (OPROST) > a-ethyl sterigmatocystin > a-methylsterigmatocystin > Sterigmatocystin> a-benzoyl sterigmatocystin (OBzST). It was apparent that the "enzyme" responsible for the conversion of the derivatives to AFB1 did not display a high degree of substrate specificity, since it was unable to recognize the difference between the various alkyl groups, either as ether or ester functional groups. An HPLC method was developed using a diode array detector. The optimum parameters for the separation of aflatoxin metabolites and the synthesised derivatives were obtained for a reversed phase Lichrosphere RP-I8 column with a 30 minute gradient elution program with water and acetonitrile as the mobile phase. Crude cell-free extracts were prepared by lyophilisation of the mycelia of A. parasiticus (Whl-11l-105) with phosphate buffer. The temperature and pH for the conversion of ST to AFB1, were found to be optimum at 28°C and 7.2, respectively. The addition of SAM (1.5 mM) and NADPH (1.5 mM) increased the conversion of ST to AFBl from 11.21 % to 27.10 %. A time course study with ST, OMST and OPROST showed that the rate of conversion to AFBl was close to linear for an incubation time of up to 60 minutes. Approximation of the reaction rate indicated a decrease in the order: OMST > ST > OPROST. This indicated that the time course reaction using whole cells was in part a measure of membrane permeability rather than substrate specificity. Molecular exclusion chromatography was used to separate enzymatic protein from primary and secondary metabolites, small biomolecules and indigenous co-factors (MW < 10 000) and the partially purified "enzyme" was concentrated by dialysis against solid sucrose. The "enzyme" was subjected to non-denaturing polyacrylamide gel electrophoresis and was found to be made of sub-units ranging from 58 kDa to over 200 kDa. Enzymatic investigations with ST, as substrate, indicated that OMST is a compulsory intermediate in the biosynthesis of AFBl. Also, enzymatic investigations of selected ST derivatives showed that the partially purified "enzyme" displayed relative specificity for these substrates, viz., OMST, OPROST and OBzST. Three xanthones, namely, 1-hydroxy-,6-dimethylxanthone, I-methoxy-3,6-dimethylxanthone and l-acetyl-3,6-dimethylxanthone were synthesised, purified and characterised spectroscopically. Whole cell studies of A. parasiticus (CMI 91019b) and A. parasiticus (Wh1-11-105) showed that these xanthones inhibited AFBl production to varying extents. Kinetic studies of cell-free extracts revealed that the 1-methoxy-3,6-dimethylxanthone derivative was a non-competitive inhibitor. The Michaelis Menten constant (Km) of approximately 5.60 uM (for OMST) was determined for a cell-free reaction at pH 7.2 and 28 QC. A Clark oxygen electrode was used to carry out oxygen consumption studies in a partially purified "enzyme" preparation. A calibration system was designed and the enzymatic conversion of OMST to AFB1 and NADPH consumption were monitored by HPLC and UV spectroscopy, respectively. From the results of these enzymatic reactions, the following stoichiometric relationship was determined: 2 mole oxygen consumed = 1 mole NADPH consumed = 1 mole AFB1 produced A tentative mechanism is discussed for the conversion of OMST to AFB1 which utilizes a monooxygenase and a dioxygenase. / Thesis (Ph.D.)-University of Natal, Durban, 1996.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.