ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluor... more ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluoroacetyl-CoA as the only substrate and a crude mitochondrial extract from Dichapetalum cymosum as enzyme source, CoASH release took place at a rate of 0.0295 μmoles/min/ mg protein, indicating the presence of a fluoroacetyl-CoA hydrolase enzyme in D. cymosum. This hydrolase could not use acetyl-CoA as a substrate. The presence of this enzyme in D. cymosum probably explains why this plant is not poisoned by its production of fluoroacetate.
ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metab... more ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metabolite, moniliformin (sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione) was isolated as the sole toxin from F. fusarioides. Isolates obtained from peanuts, sorghum, millet, peaches, soil, and dried fish were all toxic to ducklings. The chronic toxicity of an isolate from millet was determined, and 10, 25, and 50% moldy meal incorporated into commercial rat mash all proved toxic. The effect of different incubation temperatures and periods was investigated. Material produced at three different temperatures was of approximately equal toxicity, although less of the material produced at higher temperature was consumed. Analysis showed that the material incubated at 31 °C contained the most moniliformin. Self-heating to a maximum of 6-8 °C took place at all incubation temperatures. The thin-layer chromatographic analytical method was applied to four randomly selected isolates, all of which produced moniliformin, ranging in quantities from 200-840 mg/kg. Methods are described for the analyses and isolation of moniliformin from yellow corn inoculated with cultures of F. fusarioides. The analytical methodology involves either thin-layer chromatography of the extracts or purification of these on Dowex-1 (Cl-) resin; 0.2 M sodium chloride elutes the moniliformin which is subjected to quantitation by UV spectroscopy.
Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtai... more Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.
The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon d... more The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon differences in the hydrophobicity of accessible surface groups. The elution order of a group of snake venom cardiotoxins was found to vary between these two HPLC methods. Circular dichroism spectroscopy showed that the eluant acetonitrile-trifluoroacetic acid used for reversed-phase HPLC altered the conformation of the toxins, whereas the salt-buffer eluting medium used for hydrophobic-interaction HPLC did not affect toxin conformation. The retention times of cardiotoxins on reversed-phase HPLC are therefore influenced by their conformational instability in the eluting medium which causes partial or complete unfolding. Hydrophobic interaction is clearly the preferred method with which to correlate the "surface hydrophobicity" of cardiotoxins and their biological effects.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1978
... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENO... more ... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENOM FRITZ HH CARLSSON * and ABRAHAM I. LOUW ... Phospholipase A2 (Naja mossambica mos mbica fraction CM-II, [12]) was a kind gift from Dr FJ Joubert ...
ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluor... more ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluoroacetyl-CoA as the only substrate and a crude mitochondrial extract from Dichapetalum cymosum as enzyme source, CoASH release took place at a rate of 0.0295 μmoles/min/ mg protein, indicating the presence of a fluoroacetyl-CoA hydrolase enzyme in D. cymosum. This hydrolase could not use acetyl-CoA as a substrate. The presence of this enzyme in D. cymosum probably explains why this plant is not poisoned by its production of fluoroacetate.
ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metab... more ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metabolite, moniliformin (sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione) was isolated as the sole toxin from F. fusarioides. Isolates obtained from peanuts, sorghum, millet, peaches, soil, and dried fish were all toxic to ducklings. The chronic toxicity of an isolate from millet was determined, and 10, 25, and 50% moldy meal incorporated into commercial rat mash all proved toxic. The effect of different incubation temperatures and periods was investigated. Material produced at three different temperatures was of approximately equal toxicity, although less of the material produced at higher temperature was consumed. Analysis showed that the material incubated at 31 °C contained the most moniliformin. Self-heating to a maximum of 6-8 °C took place at all incubation temperatures. The thin-layer chromatographic analytical method was applied to four randomly selected isolates, all of which produced moniliformin, ranging in quantities from 200-840 mg/kg. Methods are described for the analyses and isolation of moniliformin from yellow corn inoculated with cultures of F. fusarioides. The analytical methodology involves either thin-layer chromatography of the extracts or purification of these on Dowex-1 (Cl-) resin; 0.2 M sodium chloride elutes the moniliformin which is subjected to quantitation by UV spectroscopy.
Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtai... more Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.
The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon d... more The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon differences in the hydrophobicity of accessible surface groups. The elution order of a group of snake venom cardiotoxins was found to vary between these two HPLC methods. Circular dichroism spectroscopy showed that the eluant acetonitrile-trifluoroacetic acid used for reversed-phase HPLC altered the conformation of the toxins, whereas the salt-buffer eluting medium used for hydrophobic-interaction HPLC did not affect toxin conformation. The retention times of cardiotoxins on reversed-phase HPLC are therefore influenced by their conformational instability in the eluting medium which causes partial or complete unfolding. Hydrophobic interaction is clearly the preferred method with which to correlate the "surface hydrophobicity" of cardiotoxins and their biological effects.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1978
... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENO... more ... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENOM FRITZ HH CARLSSON * and ABRAHAM I. LOUW ... Phospholipase A2 (Naja mossambica mos mbica fraction CM-II, [12]) was a kind gift from Dr FJ Joubert ...
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