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.
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