Anne-Christine Dianoux

Partial proteolysis of the natural ATPase inhibitor from beef heart mitochondria: Isolation and characterization of an active cleavage product

The activity of the mitochondrial ATPase (F r) is controlled by a small peptide (M, 10 000) referred as the natural ATPase inhibitor (IF r) [l-3]. IF, interacts with the P-subunit of F r; the binding of one molecule of IF, to one molecule of F, is sufficient to bring about full inhibition of the ATPase activity despite Fr possessing 2 or perhaps 3 P-subunits [4]. Progress in the understanding of the interaction of F, with IF, requires detailed knowledge of the structure of the two interacting peptides. We have thus undertaken to determine the amino acid sequence of IF, purified from beef heart mitochondria. Here, we describe the limited proteolytic fragmentation of IF, and the isolation in pure state of tryptic and chymotryptic fragments. Partial proteolysis by trypsin resulted in the accumulation of a tryptic peptide (T r) ofM, 8000, still capable of inhibiting the ATPase activity of F r. However, partial proteolysis by chymotrypsin resulted in a peptide (C,) of Mr 7000, which was devoid of inhibitory activity. Both Tr and Cr shared with IF 1 the carboxyl-terminal amino acids. The T, peptide differed from C, by the presence of about a dozen amino acids at the amino-terminus of T r; this short sequence, reported here, appears to be essential for the inhibitory activity of T, either directly or

Inhibition of protein kinase C from polymorphonuclear neutrophils by long chain acyl coenzyme A and counteraction by Mg-ATP

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) from bovine polymorphonuclear neutrophils was inhibited by micromolar amounts of long chain acyl-CoAs. The extent of inhibition at a given concentration of the acyl-CoAs depended on the length of the chain. A chain length of at least 12C was required for inhibition. Inhibition of protein kinase C activity was counteracted specifically by Mg-ATP.

Mass spectral identification of the blocked N-terminal tryptic peptide of the ATPase inhibitor from beef heart mitochondria

The presence of a formyl blocking group at the N‐terminus of the ATPase inhibitor has been identified and the partial sequence of the N‐terminal peptide has been determined by fast atom bombardment and field desorption coupled to mass spectrometry. Minor discrepancies in amino acid sequence of the inhibitor between the present and published data [(1981) Proc. Natl. Acad. Sci. USA 78, 7403‐7407] are reported and its relationships with other inhbitors are briefly discussed.

Effects of proteolytic fragmentations on the activity of the mitochondrial natural ATPase inhibitor

The so-called natural ATPase inhibitor, IFi, is a peptide of low M, (10 000 [1,2]), which has been purified from mitochondria of a number of species [3-81. Under appropriate conditions (slightly acidic pH, Mg-ATP), IFi binds to Fi, the hydrophilic part of the ATPase complex, and blocks its hydrolytic activity [ 1,3,9 JO]. As typically illustrated for beef heart Fi-ATPase, the inhibitory activity of IF1 is associated with its specific binding to the fl subunit of Fi [ 11 ,121. Further, although each mole of Fi contains 2 or possibly 3 fi subunits, the binding of 1 IFi to only 1 p subunit is sufficient to promote full inhibition of the hydrolytic activity of Fi [ 111. Other structural aspects concerning especially the interaction of IFi with the /3 subunit of Fi are virtually lacking. In [ 131 tryptic fragmentation of IFi close to the N-terminus yielded a peptide of 8000 MI, deprived of the N-terminal amino acid sequence, which is still capable of interacting with Fi and bringing about inhibition of the hydrolytic activity. This peptide referred to as Ti contains the same C-terminus as IFi. of the transient 8000 &f, fragment and its subsequent conversion into the 75004 peptide. The peptide bond cleavages yielding fragments Ti and Tz were identified, and the amino acid sequence around these sites of cleavage reported here. Among other proteolytic enzymes that were investigated, clostripain and thrombin were found to generate from IF i, a peptide of 8000 Mr, active as ATPase inhibitor and probably identical to Ti. Chymotrypsin and StaphyZococcus aureus V8 protease which attack IF 1 to remove larger peptide fragments than trypsin, clostripain or thrombin yielded inactive peptides. Removal of amino acids from the C-terminus of IFi by carboxypeptidase P resulted in some loss of enzymatic activity. In summary, the entire molecule of IF, is not necessary for functioning as ATPase inhibitor. In fact, a substantial sequence of amino acids corresponding to 2500 J4, can be removed from the N-terminus of IFi without loss of inhibitory activity; the other regions of the molecule are more critical for activity.

Immunological studies on the beef heart natural ATPase inhibitor: Localization of an antigenic determinant in the inhibitor molecule

Antibodies were raised against the beef heart mitochondrial ATPase inhibitor. This antiserum prevented the ability of the ATPase inhibitor to inhibit the F1 ATPase activity. Peptide fragments obtained by enzymatic cleavage of the inhibitor protein were tested by immunoblotting or ELISA for their response to the anti-inhibitor antiserum. An antigenic determinant was located in the sequence spanning His 48 to Lys 58 of the inhibitor molecule.

Specific fragmentation of natural inhibitor of mitochondrial ATPase by thrombin.

Abstract Cleavage of natural inhibitor of mitochondrial ATPase by thrombin occurs at two specific sites. First an Arg-Ser bond is split giving two peptides. The main peptide which retains integral biological activity is further cleaved at two successive Arg-Ala bonds, none of the products is able to inhibit ATPase. The isolation of these peptides and their characterization are described.

[70] Modifiers of F1-ATPases and associated peptides

Publisher Summary This chapter describes the modifiers of F1-ATPases and the associated peptides. The chemical modifiers of proteins can be divided into two classes—the group-directed reagents (chemical reagents capable of binding to side-chain residues in the investigated protein); and the active site-directed reagents (affinity and photoaffinity labels). The mitochondrial, bacterial, and chloroplast ATPases are H + -dependent ATPases (ATP synthases), as they catalyze both ATP hydrolysis and synthesis. Dicyclohexylcarbodiimide (DCCD) is used as a chemical reagent to block the proton conduction in the F 0 channel of the ATPase complex at the level of the DCCD-binding protein. Intermolecular cross-linking by random collisions between independent F 1 molecules is not predominant at the protein concentrations used for chemical modification. Intramolecular cross-linking can establish bridges in a single subunit (hairpin formation) or between two adjacent subunits but the cross-linking requires higher concentrations of reagents than inactivation.

Superoxide anion measurement by sulfonated phenyl isothiocyanate cytochrome C

Sulfonated phenyl isothiocyanate cytochrome c is suggested as a new scavenger for superoxide anion. The efficiency of the modified cytochrome c in measurements is compared with that of phenyl isothiocyanate cytochrome c and acetylated cytochrome c. Sulfonated phenyl isothiocyanate cytochrome c is water and salt soluble. Autooxidability of the pigment is not observed. The primary advantage of sulfonate phenyl isothiocyanate cytochrome c is that it appears to be specifically reduced by O2 radicals without interferences by other reactions in complex biological systems.

HPLC purification of the natural ATPase inhibitor from the yeast Candida utilis

A rapid method of preparation of the natural ATPase inhibitor (IF1) from the mitochondria of the yeast Candida utilis has been developed. It involved high performance liquid chromatography (HPLC) as the final step of purification. An active form of Candida utilis IF1 was obtained free of contaminant. Its properties are compared with those of IF1 from other sources.