K. Schray - Academia.edu (original) (raw)
Papers by K. Schray
Journal of Biological Chemistry, 1972
Abstract The interaction of Mn2+ with d-xylose isomerase from two species of bacteria has been st... more Abstract The interaction of Mn2+ with d-xylose isomerase from two species of bacteria has been studied by electron paramagnetic resonance and by the longitudinal proton relaxation rate of water solutions of the enzyme. Binary enzyme-Mn2+ complexes are detected with dissociation constants in order of magnitude agreement with kinetically determined activator constants. A 4-fold enhancement of the effect of d-xylose isomerase-Mn2+ on the proton relaxation rate of water was observed. This is decreased by substrates and inhibitors consistent with the replacement of water ligands of the enzyme-bound manganese by substrate oxygen functions. The dissociation constants of the ternary complexes of substrates and inhibitors are in agreement with their respective Km or Ki values. Differences in the enhancements of the ternary complexes of substrates and inhibitors indicate that α-anomeric forms have a greater effect than corresponding β-anomeric forms.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(lI)... more The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(lI), manganese(III) and zinc(lI) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3-10 7, 1.7-10 6 and 2.3.10 s, respectively; the manganese(Ill) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(ll) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(Ill) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.
Journal of College …, 2009
Abstract: Peer-led team learning (PLTL) has been widely adopted for enhanced learning in a variet... more Abstract: Peer-led team learning (PLTL) has been widely adopted for enhanced learning in a variety of disciplines, mostly in introductory chemistry, but also in organic chemistry, as in this study (Tien, Roth, and Kampmeier 2002). This pedagogical approach forms student ...
Analytical Chemistry, 1988
Assays for avldln and blotln are presented whlch comblne high sendtlvlty with short assay tlmes. ... more Assays for avldln and blotln are presented whlch comblne high sendtlvlty with short assay tlmes. Mlnlmum detectable concentrations are 5 ng/mL avldln and 100 pg/mL Moth and assay thne Is under 10 mln. The fluorescence polarlzatlon of a blotln-fluorescein conjugate Is monitored. Polarlzatlon varies as a functh of avidin concentration and, at ftxed avldln levels, as a functlon of competing blotln concentrations. This slmple, r a w assay has a detecth llmn about IOOO-fokl lower than the commonly used 4-hydroxyazobenzene-2tarboxyllc acid dye blndlng method. The use of biotin and avidin as a tool in biochemistry, microbiology, and immunochemistry (1-4) has experienced a very rapid growth. This interest together with biotin's role as an essential component in human biochemistry (5) has spurred the creation of a significant number of assay methods (6-22) including microbiological, colorimetric, enzymatic, radiometric, electrochemical, and fluorescent approaches. The most widely used method is that of Green (7, 8) which is a rapid, facile spectrophotometric procedure. In general, a lower detection limit has been obtained at a cost of considerably more complex and time-consuming techniques. The assay reported here is a fluorescence polarization assay relying on the use of a biotin-fluorescein conjugate. A combination of lower detection limit and a simple, rapid protocol makes this assay a good candidate for wider use.
Journal of Biological Chemistry, Mar 25, 1973
Phosphomannose isomerase is highly specific for the /3 anomer of mannose 6-phosphate. The a anome... more Phosphomannose isomerase is highly specific for the /3 anomer of mannose 6-phosphate. The a anomer has no activity as a substrate and is, at best, a poor inhibitor. This specificity agrees with previous predictions based on stereochemical arguments. In addition, phosphomannose isomerase differs from phosphoglucose isomerase, which can also catalyze the function of anomerization of its substrates. Using the specificity of phosphomannose isomerase as a test system, it is shown that yeast phosphoglucose isomerase also catalyzes the conversion of oc-mannose 6-P to p-mannose 6-P.
Journal of Biological Chemistry, Feb 10, 1974
The preferred configuration of the active substrate for rabbit liver fructose 1,6-diphosphatase h... more The preferred configuration of the active substrate for rabbit liver fructose 1,6-diphosphatase has been determined by techniques based on rapid quench kinetics to be the a! anomer of fructose-l, 6-P*. Utilization of the p anomer, however, is also catalyzed by the enzyme with a rate coefficient 5-to lo-fold less than that for the cy anomer.
Journal of Biological Chemistry, Oct 10, 1974
Archives of biochemistry and biophysics, 1981
Cancer research, 1987
Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody d... more Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen as a method for targeting radiosensitizing agents. This attachment was accomplished by the mixed anhydride method using the hemisuccinate derivative of misonidazole. Evaluation of conjugates in vitro shows a loss of antibody binding activity with increasing loading levels; however, significant binding activity is retained even at relatively high sensitizer/antibody ratios. This observation was consistent in three binding assays: a competitive radioimmunoassay; an enzyme immunoassay; and an affinity column assay. From these studies, it was concluded that the optimal loading factor for misonidazole-antibody conjugates, when it is used for immunochemotherapy lies between 8 and 15. In vitro release studies indicated that conjugates are hydrolytically stable (t1/2 = 4 days) under physiological conditions.
Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) wit... more Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypoth...
Journal of Biological …, 1973
Yeast phosphoglucose isomerase is shown to use and produce both the u and the fi anomeric forms o... more Yeast phosphoglucose isomerase is shown to use and produce both the u and the fi anomeric forms of fructofuranose 6-phosphate. Rapid quench techniques show a Z&fold preference in the utilization of the a! anomer over the /3 anomeric form. The previous report that the (Y anomer of glucopyranose-6-P
Analytical Chemistry, 1988
problems associated with eicosanoid immunoassay (20); however, only a few examples exist (21-24) ... more problems associated with eicosanoid immunoassay (20); however, only a few examples exist (21-24) and none of these are for leukotrienes. Our results indicate that LTBl can be determined by a specific RIA under circumstances where substantial amounts of regio-or stereoisomeric dihydroxyeicosatetraenoic acids or LTB4 metabolites accompany its formation. The provisional specificity of our RIA for LTB4, based on cross-reactivity values, equals or exceeds that reported by others (6-9). However, we regard the verification by NCI-GC/MS as a more rigorous demonstration of specificity and accuracy. LITERATURE CITED (1) Bray, M.
The Journal of biological chemistry, Jan 10, 1975
The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the com... more The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-x...
The Journal of biological chemistry, Jan 10, 1975
From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructos... more From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructose bisphosphate aldolase catalyzes the cleavage of beta-D-fructose 1,6-bisphosphate but not that of the alpha anomer, although the alpha anomer may be tightly bound. Yeast D-fructose bisphosphate aldolase appears to utilize both alpha and beta anomers of the substrate, with yeast apoaldolase catalyzing the interconversion of the alpha and beta forms.
The Journal of biological chemistry, Jan 25, 1973
Journal of Immunoassay, 1981
Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) a... more Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) antiserum was studied to determine conditions of maximum sensitivity. The competitive binding assay utilized showed a large increase in sensitivity with immobilized antigen levels below the levels giving rise to the maximum in the coating-antigen dilution series. The effects of reversible antigen binding to the solid phase were investigated by comparison of untreated polystyrene tubes, polystyrene tubes treated with glutaraldehyde and glass tubes activated with an aminosilane. The use of glutaraldehyde treated tubes reduced, and the use of activated glass tubes prevented the time dependent release of immobilized antigen seen with the untreated polystyrene tubes. By comparison of these solid phases, it is shown that reversible antigen immobilized in a competitive binding assay gives rise to poorer conjugate binding (three-fold), and poorer sensitivity (six-fold). A noncompetitive response was found to occur at high free antibody levels and low competing antigen concentrations. This binding behavior is moderated by the minimization of the reversible antigen immobilization.
Advances in Enzymology - and Related Areas of Molecular Biology, 1976
Transition States of Biochemical Processes, 1978
Journal of Biological Chemistry, 1972
Abstract The interaction of Mn2+ with d-xylose isomerase from two species of bacteria has been st... more Abstract The interaction of Mn2+ with d-xylose isomerase from two species of bacteria has been studied by electron paramagnetic resonance and by the longitudinal proton relaxation rate of water solutions of the enzyme. Binary enzyme-Mn2+ complexes are detected with dissociation constants in order of magnitude agreement with kinetically determined activator constants. A 4-fold enhancement of the effect of d-xylose isomerase-Mn2+ on the proton relaxation rate of water was observed. This is decreased by substrates and inhibitors consistent with the replacement of water ligands of the enzyme-bound manganese by substrate oxygen functions. The dissociation constants of the ternary complexes of substrates and inhibitors are in agreement with their respective Km or Ki values. Differences in the enhancements of the ternary complexes of substrates and inhibitors indicate that α-anomeric forms have a greater effect than corresponding β-anomeric forms.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(lI)... more The interactions of the free base porphyrin, tetra-(4N-methylpyridyl)porphyrin and its copper(lI), manganese(III) and zinc(lI) complexes with brewer's yeast type V phenylalaninyl tRNA were evaluated by UV-visible spectroscopy, circular dichroism and melting temperature studies over a range of magnesium ion concentrations and ionic strengths. Scatchard analysis of absorption spectra of the porphyrins in the presence of tRNA showed the free base, copper and zinc porphyrins to have binding constants of 7.3-10 7, 1.7-10 6 and 2.3.10 s, respectively; the manganese(Ill) complex did not demonstrate changes in its electronic spectra that enable the calculation of a binding constant. The results of the spectroscopic studies indicate a mode of binding for the free base, copper(ll) and zinc(II) complexes that is neither intercalative nor simply outside electrostatic. The magnitude of the binding constants and the UV-visible results support intercalation, but the analyses of the thermal denaturation studies and the circular dichroism evaluations suggest that the porphyrins are associating at a single site in a fold of the tertiary structure of the tRNA close to several crucial hydrogen bonds, perhaps in the vicinity of the P10 loop. That the manganese(Ill) complex does not bind in this site points to constraints on the axial thickness of a molecule that may be accommodated in this locus.
Journal of College …, 2009
Abstract: Peer-led team learning (PLTL) has been widely adopted for enhanced learning in a variet... more Abstract: Peer-led team learning (PLTL) has been widely adopted for enhanced learning in a variety of disciplines, mostly in introductory chemistry, but also in organic chemistry, as in this study (Tien, Roth, and Kampmeier 2002). This pedagogical approach forms student ...
Analytical Chemistry, 1988
Assays for avldln and blotln are presented whlch comblne high sendtlvlty with short assay tlmes. ... more Assays for avldln and blotln are presented whlch comblne high sendtlvlty with short assay tlmes. Mlnlmum detectable concentrations are 5 ng/mL avldln and 100 pg/mL Moth and assay thne Is under 10 mln. The fluorescence polarlzatlon of a blotln-fluorescein conjugate Is monitored. Polarlzatlon varies as a functh of avidin concentration and, at ftxed avldln levels, as a functlon of competing blotln concentrations. This slmple, r a w assay has a detecth llmn about IOOO-fokl lower than the commonly used 4-hydroxyazobenzene-2tarboxyllc acid dye blndlng method. The use of biotin and avidin as a tool in biochemistry, microbiology, and immunochemistry (1-4) has experienced a very rapid growth. This interest together with biotin's role as an essential component in human biochemistry (5) has spurred the creation of a significant number of assay methods (6-22) including microbiological, colorimetric, enzymatic, radiometric, electrochemical, and fluorescent approaches. The most widely used method is that of Green (7, 8) which is a rapid, facile spectrophotometric procedure. In general, a lower detection limit has been obtained at a cost of considerably more complex and time-consuming techniques. The assay reported here is a fluorescence polarization assay relying on the use of a biotin-fluorescein conjugate. A combination of lower detection limit and a simple, rapid protocol makes this assay a good candidate for wider use.
Journal of Biological Chemistry, Mar 25, 1973
Phosphomannose isomerase is highly specific for the /3 anomer of mannose 6-phosphate. The a anome... more Phosphomannose isomerase is highly specific for the /3 anomer of mannose 6-phosphate. The a anomer has no activity as a substrate and is, at best, a poor inhibitor. This specificity agrees with previous predictions based on stereochemical arguments. In addition, phosphomannose isomerase differs from phosphoglucose isomerase, which can also catalyze the function of anomerization of its substrates. Using the specificity of phosphomannose isomerase as a test system, it is shown that yeast phosphoglucose isomerase also catalyzes the conversion of oc-mannose 6-P to p-mannose 6-P.
Journal of Biological Chemistry, Feb 10, 1974
The preferred configuration of the active substrate for rabbit liver fructose 1,6-diphosphatase h... more The preferred configuration of the active substrate for rabbit liver fructose 1,6-diphosphatase has been determined by techniques based on rapid quench kinetics to be the a! anomer of fructose-l, 6-P*. Utilization of the p anomer, however, is also catalyzed by the enzyme with a rate coefficient 5-to lo-fold less than that for the cy anomer.
Journal of Biological Chemistry, Oct 10, 1974
Archives of biochemistry and biophysics, 1981
Cancer research, 1987
Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody d... more Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen as a method for targeting radiosensitizing agents. This attachment was accomplished by the mixed anhydride method using the hemisuccinate derivative of misonidazole. Evaluation of conjugates in vitro shows a loss of antibody binding activity with increasing loading levels; however, significant binding activity is retained even at relatively high sensitizer/antibody ratios. This observation was consistent in three binding assays: a competitive radioimmunoassay; an enzyme immunoassay; and an affinity column assay. From these studies, it was concluded that the optimal loading factor for misonidazole-antibody conjugates, when it is used for immunochemotherapy lies between 8 and 15. In vitro release studies indicated that conjugates are hydrolytically stable (t1/2 = 4 days) under physiological conditions.
Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) wit... more Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypoth...
Journal of Biological …, 1973
Yeast phosphoglucose isomerase is shown to use and produce both the u and the fi anomeric forms o... more Yeast phosphoglucose isomerase is shown to use and produce both the u and the fi anomeric forms of fructofuranose 6-phosphate. Rapid quench techniques show a Z&fold preference in the utilization of the a! anomer over the /3 anomeric form. The previous report that the (Y anomer of glucopyranose-6-P
Analytical Chemistry, 1988
problems associated with eicosanoid immunoassay (20); however, only a few examples exist (21-24) ... more problems associated with eicosanoid immunoassay (20); however, only a few examples exist (21-24) and none of these are for leukotrienes. Our results indicate that LTBl can be determined by a specific RIA under circumstances where substantial amounts of regio-or stereoisomeric dihydroxyeicosatetraenoic acids or LTB4 metabolites accompany its formation. The provisional specificity of our RIA for LTB4, based on cross-reactivity values, equals or exceeds that reported by others (6-9). However, we regard the verification by NCI-GC/MS as a more rigorous demonstration of specificity and accuracy. LITERATURE CITED (1) Bray, M.
The Journal of biological chemistry, Jan 10, 1975
The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the com... more The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-x...
The Journal of biological chemistry, Jan 10, 1975
From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructos... more From a series of rapid quench kinetic experiments, it has been demonstrated that muscle D-fructose bisphosphate aldolase catalyzes the cleavage of beta-D-fructose 1,6-bisphosphate but not that of the alpha anomer, although the alpha anomer may be tightly bound. Yeast D-fructose bisphosphate aldolase appears to utilize both alpha and beta anomers of the substrate, with yeast apoaldolase catalyzing the interconversion of the alpha and beta forms.
The Journal of biological chemistry, Jan 25, 1973
Journal of Immunoassay, 1981
Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) a... more Coated tube enzyme immunoassay using alkaline phosphatase conjugated to rabbit (anti-human IgG) antiserum was studied to determine conditions of maximum sensitivity. The competitive binding assay utilized showed a large increase in sensitivity with immobilized antigen levels below the levels giving rise to the maximum in the coating-antigen dilution series. The effects of reversible antigen binding to the solid phase were investigated by comparison of untreated polystyrene tubes, polystyrene tubes treated with glutaraldehyde and glass tubes activated with an aminosilane. The use of glutaraldehyde treated tubes reduced, and the use of activated glass tubes prevented the time dependent release of immobilized antigen seen with the untreated polystyrene tubes. By comparison of these solid phases, it is shown that reversible antigen immobilized in a competitive binding assay gives rise to poorer conjugate binding (three-fold), and poorer sensitivity (six-fold). A noncompetitive response was found to occur at high free antibody levels and low competing antigen concentrations. This binding behavior is moderated by the minimization of the reversible antigen immobilization.
Advances in Enzymology - and Related Areas of Molecular Biology, 1976
Transition States of Biochemical Processes, 1978