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Chemical modification of essential amino acid moieties associated with the red beet (Beta vulgaris, L.) plasma membrane hydrogen atom ATPase

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Title: Chemical modification of essential amino acid moieties associated with the red beet (Beta vulgaris, L.) plasma membrane hydrogen atom ATPase
Author(s): Gildensoph, Lynne Helen
Doctoral Committee Chair(s): Briskin, Donald P.
Department / Program: Agronomy
Discipline: Agronomy
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Agriculture, Agronomy Chemistry, Biochemistry Biology, Plant Physiology
Abstract: An investigation of essential amino acid moieties associated with the plasma membrane H$\sp+$-ATPase of red beet was undertaken using specific chemical modification. Lysine residues were modified using fluorescein-5$\sp\prime$-isothiocyanate at alkaline pH. Micromolar concentrations of this reagent during incubation of the plant ATPase at pH 9.2 resulted in enzyme inactivation. Activity was protected by the presence of the substrate, ATP or the product, ADP. This suggested an active site location for this lysine residue, consistent with data obtained with other E$\sb1$E$\sb2$-type ATPases.Phenylglyoxyl and 2,3-butanedione were used to determine if essential arginine moieties were associated with the red beet H$\sp+$-ATPase. Inhibition kinetics for both reagents at millimolar concentrations revealed the presence of one essential arginine. Inclusion of either ATP or ADP in the incubation medium protected the enzyme against inactivation by both reagents. Treatment of the H$\sp+$-ATPase with phenylglyoxyl inhibited phosphoenzyme formation. However, it was uncertain whether this represented an effect on ATP binding or the production of the covalent aspartyl intermediate. Based upon the results of this study, it was proposed that the H$\sp+$-ATPase contains an active site arginine residue which might interact with the phosphate groups of the ATP substrate.Transient state kinetic studies have suggested the presence of an essential histidine moiety in the red beet plasma membrane H$\sp+$-ATPase. The histidine modifying reagent diethylpyrocarbonate was used to investigate this possibility. This reagent was an effective inhibitor of the ATPase in the micromolar concentration range and ATP was only partially effective in protecting against inhibition. Thus, this essential moiety may instead reside in a separate conformationally active region of the protein. Phosphoenzyme formation and discharge by unlabeled ATP addition were unaffected by treatment with this reagent. This was consistent with diethylpyrocarbonate acting to block the catalytic cycle at the level of the first phosphoenzyme intermediate (E$\sb1$P).Characterization of essential amino acid moieties associated with the plant plasma membrane H$\sp+$-ATPase represents an important step towards understanding the mechanism of this protein. These results will complement recent information regarding deduced amino acid sequences for this enzyme obtained using gene cloning methodology.
Issue Date: 1989
Type: Text
Language: English
URI: http://hdl.handle.net/2142/22343
Rights Information: Copyright 1989 Gildensoph, Lynne Helen
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9010864
OCLC Identifier: (UMI)AAI9010864
 

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