Subunit dissociation of Busycon canaliculatum hemocyanin

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of manganese(II) and small ligands with Busycon canaliculatum hemocyanin

Cyanide (5 X 10(-3) M) and thioacetamide (5 X 10(-3) M) increase the P50 values (P02 required for 50% oxygenation) of hemocyanin by 100%, respectively. Using an ion-exchange method involving 14CN-, we have found that cyanide forms a 1:1 complex with hemocyanin in the concentration range examined: Kf = 2.3 X Mw M-1 at room temperature, where Kf is association constant and Mw is molecular weight of hemocyanin. This strong binding of cyanide to hemocyanin is to be expected from the effect of this ion on the oxygenation of hemocyanin. The effects of manganese(II) ion and fluoride on the oxygenation of hemocyanin are found to be weak. The nmr measurements, however, suggest that manganese(II) ion does have some interactions with the active site of hemocyanin.     

Spectroscopic studies of ligand perturbation effects on the half oxidized action site of Busycon canaliculatum hemocyanin.

We report the preparation and characterization of an important new series of hemocyanin derivatives containing a variety of anions bound to the cupric ion in a singly oxidized active site. Spectroscopic studies on these half met-L derivatives, Cu(I)…Cu(II)-L, where L = CN^?, NO₂^?, CH₃CO₂^?, and N₃^? reveal that the unpaired electron is localized on one copper in an approximately tetragonal site with a d_{x² ? y²} ground state. Ligand variation is used as a perturbation on the spectral features to show that the small molecule (and by analogy, oxygen in oxyhemocyanin) is bound in the equatorial plane of the copper.     

Ultrastructure of the radula protractor of Busycon canaliculatum

Summary The distribution of the sarcoplasmic reticulum and sarcolemmic tubules in the radula protractor muscle of the whelk, Busycon canaliculatum, has been investigated. The sarcoplasmic reticulum consists of an interconnected system of cisternae and tubular channels. The cisternae are closely associated with the sarcolemma. The tubular channels project from the cisternae into the interior of the cell and run parallel to the long axis of the myofilaments. Parallel tubular channels are interconnected with one another by short branches. This finding of an elaborate sarcoplasmic reticulum supports previous physiological work on this smooth muscle which indicated the presence of an intracellular compartmentalization of calcium ions. There is also an extensive system of tubular invaginations of the sarcolemma which we have termed sarcolemmic tubules. These tubules are 600 Å in diameter and about 0.5 microns in length. There is a substructure associated with the leaflet of the tubular membrane bordering the extracellular space. The sarcolemmic tubules penetrate only half a micron from the surface of the cell and interdigitate with the sarcoplasmic reticulum associated with the sarcolemma. Calculations have shown that the surface area of this smooth muscle cell is more than doubled by the presence of sarcolemmic tubules.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Molecular weights of aggregation states of Busycon hemocyanin.

Molecular weights of all hemocyanin aggregates which can be homogeneously isolated have been measured by sedimentation equilibrium. The larger aggregates, which are the ones present under physiological conditions, are, to a very close approximation, integral multiples of a 4.4 x 10(6)-dalton, 60 S species. Dissociation of the 60 S species at high pH gives heterogeneous samples in which the smallest species has a molecular weight of 300,000. The smallest subunit which can be produced in denaturing solvents also has a molecular weight of 300,000.     

Oxygen binding to hemocyanin: a resonance Raman spectroscopic study

Oxygenation of hemocyanin gives rise to resonance Raman peaks at 742 and 282 cm^?1. The 742 cm^?1 peak which is in resonance with the 575 nm charge transfer band shifts to 704 cm^?1 when ¹⁸O₂ is substituted for ¹⁶O₂. Our results establish that the bound oxygen is in the form of peroxide (O₂^2?). The 282 cm^?1 peak which is in resonance with the 340 nm optical transition is insensitive to isotopic substitution, suggesting that the 282 cm^?1 peak corresponds to a vibration involving the magnetically-coupled Cu(II)··Cu(II) centers.     

Effects of monensin on the response to low potassium in a molluscan muscle, the radular protractor of Busycon canaliculatum

1. A sucrose gap technique was used to study the effect of a sodium ionophore on the potential changes which occur during superfusion with potassium-free solution. Crucial values were checked with a microelectrode technique. 2. Potassium-free solution induces a complex response consisting of a hyperpolarizing phase (HP) and then a depolarization phase (DP) during exposure to zero K+, followed by a transient extra hyperpolarizing phase (EHP) on readmission of K+. 3. The sodium ionophore, monensin, has the effect of increasing the amplitude of the DP, in perfusion medium of normal Na+ content. This is similar to the effect of treatment with neurohumors and to the after-effect of direct electrical stimulation. The effects of ACh and monensin are additive. 5. These effects are consistent with an action of monensin in increasing Na+ flux in the direction of the concentration gradient and support the hypothesis that neurohumors stimulate the sodium pump by increasing Na+ influx.     

The action of RFamide neuropeptides on molluscs, with special reference to the gastropods Buccinum undatum and Busycon canaliculatum

The ever-growing RFamide neuropeptide superfamily has members in all animal phyla. Their effects in molluscs, on both smooth and cardiac muscle as well as on neurons, has been studied in detail. These neuropeptides exert a variety of functions: excitatory, inhibitory or even biphasic. Firstly, the literature on the excitatory effect of the RFamide neuropeptides on molluscan muscle and neurons has been reviewed, with greater emphasis and examples from the gastropods Buccinum undatum and Busycon canaliculatum. The peptides seem to be potent activators of contraction, sometimes generating slow tonic force and other times twitch activity. Secondly, the literature on the inhibitory effect of the superfamily has been reviewed. These peptides can exert an inhibitory effect, hyperpolarizing the cells rather than depolarizing them. Thirdly, the neuropeptides may play a variety of other roles, such as contributing to the regulation or maturation process of the animals. There have been cases recorded of RFamide neuropeptides acting as potent venoms in members of the Conus sp. The pathway of action of these multiple roles, their interaction with the parent neurotransmitters acetylcholine and serotonin, as well as the calcium dependency of the RFamide neuropeptides has been discussed, again with special reference to the above mentioned gastropods. A better understanding of the mode of action, the effects, and the importance of the RFamide neuropeptides on molluscan physiology and pharmacology has been attempted by reviewing the existing literature, recognizing the importance of the RFamide neuropeptide actions on molluscs.     

A light-scattering study of the effect of calcium chloride on the molecular weight of Busycon hemocyanin.

Solutions of Busycon canaliculatum have been studied by light scattering. In 0.05 M Trizma buffer +0.1 M NaCl at pH 7.0 at 14 degrees, the weight-average molecular weight is 8.9 X 10(6). In the presence of added CaCl2 (0.02 M), the molecular weight of the protein increases to 10.7 X 10(6), and the second virial coefficient is reduced. At pH 9.95, the molecular weights with and without 0.02 M CaCl2, are 3.7 X 10(6) and 1.3 X 10(6), respectively; and the effect of Ca++ in reducing the second virial coefficient is much greater than at pH 7.0. These results can be understood on the basis that at pH 7.0, ca++ increases the association of hemocyanin, by binding and intermolecular linkage through the carboxyl groups of protein side chains. At pH 9.95, amino groups are deprotonated and therefore also become available for Ca++ binding. The relative effect of Ca++ in enhancing the association of hemocyanin therefore becomes greater at the higher pH.