Studies on Fusarium-Gladiolus Interactions

Using standardized conditions, 65 genotypes of Gladiolus were screened for Fusarium resistance. High levels were found in 'large-flowered" types, Primulinus hybrids, G. callianthus, G. garnierii , and G. dalenii. Some accessions of G. dalenii exhibited no disease symptoms when inoculated with two standard test isolates. No resistance was found in 'small-flowered' types. To estimate race-specifity of the resistance, eight highly resistant Gladiolus genotypes were tested in an in vitro test against 43 isolates of F. oxysporum f.sp. gladioli. Two isolates were able to partially infect the G. dalenii accessions and this was confirmed using whole plants. Implications for resistance breeding are discussed.     

Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins.

We report Hammett correlations, using 8-substituted flavins, to clarify the mechanism of hydroxylation by p-hydroxybenzoate hydroxylase (PHBH). The 8-position of the FAD isoalloxazine ring was chosen for modifications, because in PHBH it has minimal interactions with the protein, and it is accessible to solvent and away from the site of hydroxylation. Although two intermediates, a flavin-C4a-hydroperoxide and a flavin-C4a-hydroxide, are known to participate in hydroxylation, the mechanism of oxygen transfer remains controversial. Mechanisms as diverse as electrophilic aromatic substitution, diradical formation, and isoalloxazine ring opening have been proposed. In the studies reported here, it was possible to monitor spectrally each of the individual steps involved in hydroxylation, because the FAD cofactor acts as a reporter group. Thus, with PHBH, substituted separately with nine derivatives of FAD altered in the 8-position, quantitative structure-reactivity relationships (QSAR) have been applied to probe the mechanisms of formation of the flavin-C4a-hydroperoxide, the conversion to the flavin-C4a-hydroxide with concomitant oxygen transfer to the substrate, and the dehydration of the flavin-C4a-hydroxide to form oxidized FAD. The individual chemical steps in the mechanism of PHBH were not altered when using any of the modified flavins, and normal products were obtained; however, the rates of individual steps were affected, and depended on the electronic properties of the 8-substituent. Increased hydroxylation rates were observed when a more electrophilic flavin-C4a-hydroperoxide (i.e., with an electron-withdrawing substituent at the 8-position) is bound to PHBH. On the basis of QSAR analysis, we conclude that the mechanism of the hydroxylation step is best described by electrophilic aromatic substitution.     

Acyl chain and headgroup specificity of human plasma lecithin:cholesterol acyltransferase. Separation of matrix and molecular specificities

To determine how substrate fluidity and molecular structure independently regulate cholesteryl ester formation, the substrate specificity of lecithin:cholesterol acyltransferase with respect to a number of model reassembled high density lipoproteins (R-HDLs) is reported. The R-HDLs are composed of 1 mol % apolipoprotein A-I, 89 mol % of sphingomyelin or a nonhydrolyzable diether analog of phosphatidylcholine (PC) plus 10 mol % of test lipids that are potential acyl donors; a trace of [3H]cholesterol, which permits quantification of cholesteryl ester formation is also included. With respect to the lipid class of the acyl donor, the rate of ester formation decreases in the order phosphatidylethanolamine greater than phosphatidylcholine greater than N,N,-dimethylphosphatidylethanolamine greater than phosphatidylglycerol - phosphatidic acid greater than phosphatidylserine greater than dipalmitin greater than tripalmitin. Within an R-HDL composed of 90% PC ether or sphingomyelin, the relative rates of ester formation are greatest for dipalmitoyl and dimyristoyl PC, with distearoyl PC being almost unreactive; in a solid lipid environment, the rate with respect to unsaturation of the PC is greatest for oleate. In a fluid lipid environment, all unsaturated PCs were utilized nearly equally. All lipids tested were most reactive within an R-HDL composed of an unsaturated PC ether and least reactive within an R-HDL composed mostly of sphingomyelin. These results suggest that the rates of ester formation by lecithin:cholesterol acyltransferase are separate functions of the identity and the microscopic environment of the acyl donor. This is the first example of the use of diether analogs for the separation of the effects of macromolecular and molecular structure on the specificity of lecithin:cholesterol acyltransferase.     

Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center

Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.     

Surface activation of pro-cathepsin L.

Pro-cathepsin L is an inactive zymogen that has been shown previously to undergo autolysis at pH 3.0 to give mature forms of the enzyme. We have now been able to demonstrate that this enzyme can undergo activation at pH 5.5 in the presence of negatively charged surfaces. Activation could also be measured at pH 6.0, but no activation occurred at pH 6.5 or higher. The initiation of activation depends upon the presence of a small percentage of active pro-enzyme, and this is then followed by a more rapid activation to give mature forms of the enzyme. No significant intermediate molecular forms of the enzyme were seen. The time taken for processing of the pro-enzyme to single-chain mature enzyme is comparable to that seen in biosynthetic pulse-chase experiments.     

Kynuramine, a fluorescent substrate and probe of plasma amine oxidase

The fluorescence substrate kynuramine was used as a probe of the catalytic site of plasma amine oxidase. Under anaerobic conditions, the binding of kynuramine causes several spectroscopic changes. The Stokes shift (deltav = 5326 cm-) associated with binding of the substrate to the enzyme can be attributed to nonpolar properties of the binding site, whereas the increase in emission anisotropy (A = 33) indicates rigid attachment of the substrate to the enzyme. The fluorescence enhancement that follows the binding of substrate was used to determine the association constant (Ka). The enzyme plasma amine oxidase binds only 1 molecule of substrate with a Ka = 1.8 X 10(5) M-1 under anaerobic conditions. The use of fluorescence substrates seems to offer the possibility of monitoring conformational changes occurring prior to the catalytic event.