In the course of evolution, Ca2+ has emerged as the most versatile intracellular messenger. Its concentration within cells is controlled by reversible binding to specific classes of proteins that act as Ca2+ sensors to decode its information before passing it on to targets. The decoding operation is based on specific conformational changes in the sensor proteins. Other proteins intrinsic to membranes simply control Ca2+ concentration without processing its message, by transporting it across membrane boundaries. They are located in the plasma membrane and in the membranes of the organelles (the endo(sarco)plasmic reticulum, the mitochondria, the nuclear envelope), which play distinctive roles in the cellular homeostasis of Ca2+. Ca2+ is an ambivalent signaling agent. It carries information to virtually all processes important to cell life (e.g., it couples excitation to contraction, secretion, gene transcription, and controls enzyme activity through protein phosphorylation-dephosphorylation), but also transmits signals that promote the programmed demise of cells. When escaping control, Ca2+ also precipitates toxic cell death.

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca 2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca 2+ . The sensitivity of the catalytic activity of the recombinant protease to Ca 2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca 2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m-and m-calpain isoforms, intracellular Ca 2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.Keywords: baculovirus; calpain; p94; calcium.The muscle specific calpain isoform p94 (calpain 3) has attracted wide attention due to its involvement in one of the forms of limb-girdle muscular distrophy: a number of inactivating mutations of the p94 gene cause limb-girdle muscular distrophy type 2A (LGMD2A), suggesting that p94 activity is essential for normal muscle function [1]. Very little is known on the substrates of p94 in muscles in vivo, but one recent report has compellingly suggested that one of them might be IkBa, the inhibitor of the NFkB-Rel transcription factor family [2]. The domain organization of p94 repeats the canonical four-domain structure of the large subunits of the ubiquitously expressed m-and m-calpains [3], with domains II and IV corresponding to the cysteine protease and putative calcium-binding regions, respectively. The structure of p94, however, is characterized by three unique inserts [4]: a prolinerich domain (NS) of unknown function at the N-terminal end, a 62 residue sequence (IS1) between the catalytic cysteine and histidine of domain II, and a 77 residue lysine-rich sequence (IS2) that separates domains III and IV. The location of the IS1 insert suggests its involvement in the proteolytic activity of the protein. The domain has been recently identified as the target of the autocatalytic cleavage of the protease [5]. The IS2 insert contains a potential nuclear localization signal and is essential for the binding of p94 to the giant sarcomeric protein connectin [6]. Thus, it may be involved in the intracellular distribution of the protease, which has been suggested to shuttle between the nuclear and cytoplasmic compartments of muscle cells [2,6]. Finally, p94 differs from the large subunits of m-and m-calpain in it...

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The problem of whether the rate of ATP synthesis is proportional to the magnitude of the protonmotive force has been studied in submitochondrial particles. It was found that the rate of ATP synthesis can decrease at constant protonmotive force and is more closely related to the rate of substrate oxidation.The chemiosmotic mechanism (Mitchell, 1966(Mitchell, , 1976 of energy coupling requires that there should be a defined relationship between the magnitude of a transmembrane electrochemical proton gradient (protonmotive force, Ap) and the extent to which processes driven by Ap (e.g. ATP synthesis) are displaced from equilibrium. In contrast, the statement of the chemiosmotic hypothesis (Mitchell, 1966(Mitchell, , 1976 makes no prediction as to the relationships between changes in the rates of either energy-yielding or energy-utilizing processes and the magnitude of Ap, yet knowledge of these relationships is important both for an overall understanding of oxidative phosphorylation (and other related processes) as well as for applying the

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