Carp myosin rod was isolated from the chymotryptic digest of myosin by using DEAE-Toyo pearl chromatography.The carp rod thus prepared was very similar to rabbit rod in its molecular mass, viscosity, sedimentation velocity, and amino acid composition . Changes in tryptophan fluorescence intensity indicated that carp rod more easily unfolded upon addition of urea or gua nidine-HCI, and thus was structurally less stable than rabbit rod . It was found that the addition of 8-anilino-1-naphthalene sulfonate (ANS) to carp rod solution resulted in no change in fluore rescence intensity, providing no information on its structural stability . However, ANS was an excellent probe for detecting the structural change of myosin subfragment-1 (S-1) portion; namely carp S-1 required a lower urea concentration than rabbit S-1 for inducing an increase inthe ANS fluorescence intensity.

Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.

Upon heating carp myofibrils at 40 degrees C, the amount of myosin that is soluble and monomeric dropped very quickly, roughly 5 times faster than the ATPase inactivation. This rapid decrease of solubility was well explained by a rapid denaturation of the rod portion as measured by chymotryptic digestibility. Chymotryptic digestion of heated myofibrils in a low-salt medium with EDTA generated a reduced amount of rod and subfragment-1 (S-1). The decrease of S-1 produced from the heated myofibrils was consistent with the ATPase inactivation. The decrease of rod produced from the heated myofibrils was explained by the increased susceptibility of the heavy meromyosin (HMM)/light meromyosin (LMM) junction to chymotryptic. It was, therefore, concluded that the fastest event occurring in the myosin molecule upon heating of myofibrils is the irreversible exposure of the HMM/LMM junction.

Carp dorsal myosin formed oligomers that retained ATPase activity upon heating. Cleavage of the oligomeric myosin at subfragment-1 (S-1)/rod junction released monomeric S-1 and rod, indicating that ATPase retaining myosin associated near the S-1/rod junction. The digest also contained rod oligomers. Heating a mixture of S-1 and rod generated neither ATPase retaining S-1 oligomers nor rod oligomers. Electron microscopic observation of the heated myosin revealed that some oligomers were formed by associating at the S-1/rod joining region, exhibiting a recognized double head, probably ATPase retaining oligomers. No myosin oligomers associated at the tail region were observed, thus, rod aggregation would be formed at its very restricted region near the S-1/rod junction. Based on the findings, we proposed that the neck structure is important in the thermal oligomerization process of myosin.

Most molluscs have blue blood because their respiratory molecule is hemocyanin, a type-3 copper-binding protein that turns blue upon oxygen binding. Molluscan hemocyanins are huge cylindrical multimeric glycoproteins that are found freely dissolved in the hemolymph. With molecular masses ranging from 3.3 to 13.5 MDa, molluscan hemocyanins are among the largest known proteins. They form decamers or multi-decamers of 330- to 550-kDa subunits comprising more than seven paralogous functional units. Based on the organization of functional domains, they assemble to form decamers, di-decamers, and tri-decamers. Their structure has been investigated using a combination of single particle electron cryo-microsopy of the entire structure and high-resolution X-ray crystallography of the functional unit, although, the one exception is squid hemocyanin for which a crystal structure analysis of the entire molecule has been carried out. In this review, we explain the molecular characteristics of molluscan hemocyanin mainly from the structural viewpoint, in which the structure of the functional unit, architecture of the huge cylindrical multimer, relationship between the composition of the functional unit and entire tertiary structure, and possible functions of the carbohydrates are introduced. We also discuss the evolutionary implications and physiological significance of molluscan hemocyanin.

Thermal denaturation of myofibrils from various species of fish was investigated by measuring ATPase inactivation, myosin aggregation, myosin subfragment-1 (S-1) and rod denaturation rates as studied by chymotryptic digestion. Decrease in monomeric myosin (myosin aggregation) was always faster than the ATPase inactivation for all myofibrils tested. The relative denaturation rate of rod to that of S-1 differed from species to species. Preceded denaturation of rod was observed with some species, and the opposite was true with other species. The denaturation pattern was explained by the different magnitude of S-1 stabilization by F-actin in myofibrils at low salt medium. Myofibrils which receive a great stabilization by F-actin as studied by ATPase inactivation showed the preceded rod denaturation pattern, and vice versa. S-1 portion, not F-actin, determined the different stabilization of S-1 by F-actin in myofibrils.

Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 1 is a 110-kDa membrane protein and a representative member of P-type ion-transporting ATPases. SERCA1a catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4). According to the E1/E2 transport mechanism ( Fig. 1) (3-7), the enzyme is activated by the binding of two Ca 2ϩ ions (E1⅐Ca 2 , steps 1-2) and then autophosphorylated by MgATP to form an ADP-sensitive phosphoenzyme (E1P, step 3). On formation of this EP, the bound Ca 2ϩ ions are occluded in the transport sites. The subsequent isomeric transition to the ADP-insensitive form (E2P, step 4) will result in a reduction in affinity and a change in orientation of the Ca 2ϩ binding sites and thus a Ca 2ϩ release into lumen (step 5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). The main kinetic limitation in this cycle is associated with the mechanism of Ca 2ϩ release before the hydrolysis of E2P (8, 9). E2P can also be formed from P i in the presence of Mg 2ϩ and absence of Ca 2ϩ by reversal of the hydrolysis of E2P. The three-dimensional structure of Ca 2ϩ -ATPase with bound Ca 2ϩ (E1⅐Ca 2 ) and, very recently, the structure without bound Ca 2ϩ and with bound thapsigargin (E2(TG)), were solved by x-ray crystallography at the atomic level (10, 11). The enzyme has three cytoplasmic domains (A, N, and P) that are widely separated in E1⅐Ca 2 and associated in E2(TG). The modeling with a low resolution map of tubular crystals formed with decavanadate (E2V) revealed (10) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V (see Fig. 7). Our previous limited and systematic proteolysis experiments showed (12, 13) that E2V is very similar to the Ca 2ϩ -released form of E2P in the domain organization and that this EP is the intermediate with the most compactly organized headpiece in the catalytic cycle. The results further indicated that a large motion of the A domain (i.e. rotation by ϳ90° (10)) and the strong association of the A domain with the P and N domains most likely occur during the isomerization of EP and Ca 2ϩ release and suggested that the stabilization energy provided by intimate contacts between all three cytoplasmic domains in E2P provides energy for moving transmembrane helices and releases the bound Ca 2ϩ ions. To substantiate such changes in cytoplasmic domain organization and their roles in the Ca 2ϩ transport, it is essential to find the regions and residues involved in the domain-domain interactions and reveal their actual roles in the catalytic steps. The conserved outermost TGES loop (Thr 181 -Ser 184 ) on the A domain is situated at the interface of the A and P domains in E2V. This loop was previously found to be essential for the isomerization of EP (14) and predicted by iron-catalyzed cleavage with Na ϩ /K ϩ -ATPase to participate in Mg 2ϩ binding with specific residues in the conserved TGDGVND loop (starting

The effect of salt concentration on the thermal denaturation profile of myosin in walleye pollack and carp myofibrils was compared by studying the subfragment-1 (S-1) and rod denaturation rates upon heating. Species-specific denaturation mode observed at 0.1 M KCl was no longer detected when samples were heated above 0.5 M KCl, where S-1 and rod denaturation rates were identical to each other. As the heating of the chymotryptic digest of myofibril formed practically no rod aggregates, S-1 denaturation in a form of myosin was the rate limiting step for rod aggregate formation. As the aggregate formation by rod was remarkably suppressed by lowering the temperature, the free movement of myosin tail upon heating was suggested to play an important role in the rod aggregate formation in a high salt medium.