Sarcoplasmic reticulum Ca 2+ -ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca 2+ . Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P i and phosphorylated state analogues produced by the binding of F 3 or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg 505 ). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues. ß

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Arginine kinase (AK) was isolated from the longitudinal muscle of the sea cucumber Stichopus japonicus. Unlike the monomeric 40 kDa AKs from molluscs and arthropods, but like the cytoplasmic isoenzymes of vertebrate creatine kinase (CK), the Stichopus enzyme was dimeric. To explore the evolutionary origin of the dimeric AK, we determined its cDNA-derived amino acid sequence of 370 residues. A comparison of the sequence with those of other enzymes belonging to the phosphagen kinase family indicated that the entire amino acid sequence of Stichopus AK is apparently much more similar to vertebrate CKs than to all other AKs. A phylogenetic tree also strongly suggests that the Stichopus AK has evolved from CK. These results support the conclusion that AK evolved at least twice during the evolution of phosphagen kinases: first at an early stage of phosphagen kinase evolution (its descendants are molluscan and arthropod AKs) and secondly from CK later in metazoan evolution. A comparison of the amino acid sequence around the guanidino specificity (GS) region (which is a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) of the Stichopus enzyme with those of other phosphagen kinases showed that the GS region of the Stichopus enzyme was of the AK type: five amino acid deletions in the flexible loop region that might help to accommodate larger guanidine substrates in the active site. The presence of the AK-type deletions in the Stichopus AK, even though it seems that the enzyme's most immediate ancestor was probably CK, strongly suggests that the GS region has a role in substrate specificity. Stichopus AK and presumably other echinoderm AKs seem to have evolved from the CK gene; the sequence of GS region might have been replaced by the AK type via exon shuffling. The presence of an intron near the GS region in the Stichopus AK gene supports this hypothesis.

Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys 876 -Cys 888 disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca 2؉ -ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys 876 -Cys 888 disulfide bond in the expressed wild type. All the three mutants possessed high Ca 2؉ -ATPase activity. In contrast, no Ca 2؉ transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from P i . Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca 2؉ -ATPase; activation of the catalytic site upon high affinity Ca 2؉ binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADPinsensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca 2؉ from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys 876 -Cys 888 disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca 2؉ pathway.The Ca 2ϩ -ATPase of adult fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) 1 is a 994-residue membranebound protein (1, 2) that catalyzes Ca 2ϩ transport coupled to ATP hydrolysis (3, 4). In the catalytic cycle, the enzyme is activated by binding of two Ca 2ϩ ions to the transport sites from the cytoplasmic side, and then ␥-phosphoryl group of ATP is transferred to Asp 351 (5-7) to form ADP-sensitive EP, which can react with ADP to form ATP (8 -10). Upon formation of this EP, the two Ca 2ϩ ions are occluded. A subsequent rate-limiting transition of ADP-sensitive EP to ADP-insensitive EP, which cannot react with ADP, results in release of the Ca 2ϩ ions into the lumen. Finally, ADP-insensitive EP is hydrolyzed to form P i and the dephosphoenzyme. This EP can also be formed from P i in the absence of Ca 2ϩ by reversal of its hydrolysis (11, 12). The Ca 2ϩ -ATPase contains ten transmembrane-helices (M1 to M10), and the bound two Ca 2ϩ ions are shown to be located side by side near the center of four helices, M4, M5, M6, and M8 in the crystal structure (13). The ATP binding site and phosphorylation site are located on the large cytoplasmic loop between M4 and M5 (13, 14). Luminal loops are short except for the one connecting M7 and M8, the loop 7-8 (approximate...