Bromobimane crosslinking studies in oligomycin‐sensitive ATPase from beef heart mitochondria

Abstract

Using a bromobimane fluorescent label the il4,3 1000 protein band oligomycin-sensitive (OS)-ATPase from beef heart mitochondria is shown to become much intensified by 2-mercaptopropionylglycine.In the presence of 3.5 nmol/mg protein of the thiol reagent ATP-Pi exchange activity is increased by 90%. With the fluorescent crosslinking reagent dibromobimane (DB) we show that a new fluorescent peak appears between Mr 50000 and 60000. ATP-Pi exchange is very much decreased by DB. The results suggest that for regulation of ATP-synthetase activity sulfhydryl groups in the region of the I%& 31000 protein(s) play an important role. Mitochondrial OS-ATPaseBromobimane labeling Crosslinking M, 31000 protein(s)

Translocation of Loops Regulates Transport Activity of Mitochondrial ADP/ATP Carrier Deduced from Formation of a Specific Intermolecular Disulfide Bridge Catalyzed by Copper-o-Phenanthroline

“…Zimmer and co-workers (18,19) examined the effects of cross-linking reagents including Cu(OP) 2 on an oligomycin-sensitive ATPase preparation from bovine heart mitochondria and found that it modified a 31-kDa protein significantly. Later, Joshi and Torok reported that an ADP/ATP carrier contaminating the preparation of ATPase was the protein modified by Cu(OP) 2 and that Cu(OP) 2 caused intermolecular and intramolecular disulfide bridges in electron transport particles from bovine heart mitochondria (20), but it seemed to induce only intramolecular cross-linking of the carrier present in the preparation of H ϩ -ATPase (21).…”

ADP‐ and oligomycin‐sensitive redox behavior of F0 b thiol in ATPsynthase depends on neighbored primary structure: Investigations using 14‐C‐labeled alpha lipoic acid

“…As is becoming evident from the foregoing discussion, in mitochondria, oligomycin -sensitive thiol reactivity occurs to a remarkable extent [1,4,7,8,[18][19][20]33,43,57,58,61,62,64,66]. Such mitochondrial -SH reactivity can be also deduced to working rat hearts in experimentation with MPG [65] and alpha lipoic acid [67].…”

“…It was also considered very likely that the inhibition of the ATP-Pi exchange activity by NEM was due to its reaction with SH group of the 30 kDa subunit [8]. Conversely, that same subunit of 30-31 kDa was increased in staining intensity together with augmented ATP-Pi exchange by SH reagent 2-mercaptopropionylglycine (MPG) [64]. According to Gaballo et al [22] in the functional state of ATPsynthase, which could be compared to our native enzyme in the presence of ADP, there is a free SH-group at 25-27 kDa protein, the so-called PVP(F01)b subunit (m.w.…”

“…2). Our previous findings [61][62][63][64]20,21,65] using 2-mercapto-propionylglycine (MPG) and the more recent results using alpha lipoic acid [3,11,34,39,47,50,66,68], could, at least in part, become explained by reversible blockade (R-S-MPG; R-S-LA), of reactive sulfhydryl groups at strategic membrane sites including the ATPsynthase with excessive reversible reactivity of the Fo b subunit preserving mobility and structure changes during ATP synthesis.…”

Three Dimensional Structures of Proteins and Protein Complexes from Chemical Cross-Linking and Mass Spectrometry: A Biochemical and Computational Overview

“…The cross-linking reaction should be carried out in a condition that is as close as possible to the native physiological condition and the presence of the cross-linker should not appreciably distort the protein structure from its native conformation. Interestingly, most enzymes to which cross-linkers have been added tend to retain their enzymatic activity (Zimmer et al, 1982). Also, in Section 9, a number of cases where identified crosslinks were found to be in accord with the known structure, with few exceptions, were discussed.…”