Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff)

The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Hemocyanin of the chiton, Stenoplax conspicua (Dall)

1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 X 10(6) daltons, (dt) and a sedimentation coefficient of 60 S. 2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 10(5) and 4.17-4.75 x 10(5) dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly. 3. The pH dependence of the molecular weights from pH 4.5 to 11 exhibit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer. 4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins. 5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+, and analyzed by means of the decamer-dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (Napp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.     

Hemocyanin of the chiton Acanthopleura granulata

The subunit structure and solution conformation of the hemocyanin of the chiton Acanthopleura granulata were investigated by light-scattering, ultracentrifugation, viscosity, absorbance, and circular dichroism methods. The molecular weight, determined by light scattering at pH 7.4 in the presence of 0.05 M Mg2+ and 0.01 M Ca2+, was (4.2 +/- 0.3) X 10(6), while those of dissociated subunits in the presence of 8.0 M urea (at pH 7.4) and at pH 10.7 were found to be 4.57 X 10(5) and 4.58 X 10(5), respectively. Circular dichroism and absorbance measurements at 222 and 346 nm indicate only minor changes in the conformation of the folded domains of the hemocyanin subunits in these dissociating solvents. As with the hemocyanins of the snails Busycon canaliculatum, Lunatia heros, and Littorina littorea, exposure to 4.0-6.0 M guanidinium chloride (GdmCl) is found to produce unfolding of the domains, resulting in much more pronounced spectral changes and a further drop in molecular weight. A Mw of 3.2 X 10(5) was obtained with Acanthopleura hemocyanin in 6.0 M GdmCl, suggesting hidden breaks in the polypeptide chains analogous to those observed with the gastropodan hemocyanins. Both urea and pH dissociation showed gradual declines in the molecular weights, consistent with a decamer-dimer-monomer scheme of subunit dissociation. The bell-shaped molecular weight profiles obtained in the pH region from 5 to 11 can be accounted for by assuming two proton-linked groups per dimer, characterized by apparent pK values of 5.5 and 9.5, and the further involvement of five to eight acidic and five to eight basic groups per monomer, having apparent pK values of 5.0 and 10.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical studies of the hemocyanin of the marine gastropod, Kelletia kelleti (Forbes).

1. The hemocyanin of the Californian whelk, Kelletia kelleti, investigated at pH and ionic conditions close to physiological, has a molecular weight close to 9.0 x 10(6) and a sedimentation constant of 114S, characteristic of the di-decameric structure of molluscan hemocyanins. Light-scattering measurements at pH 8.0, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 9.0 +/- 0.6 x 10(6), and scanning transmission electron microscopy produced nearly the same particle mass of 9.22 +/- 0.50 x 10(6) daltons (Da). 2. Light-scattering measurements on the fully dissociated monomers in the presence of 8.0 M urea and at pHs 10.6 and 11.0 gave molecular weights of 4.50 x 10(5)-4.91 x 10(5), that are close to one-twentieth of the mass of the parent di-decameric hemocyanin assembly. 3. Changes in pH produced a bell-shaped molecular weight profile, with molecular weights close to 9.0 x 10(6) in the pH region of about 5.5-8.0, and progressive dissociation to 4.5 x 10(5) Da monomers in the region below pH 4.0 and above pH 9.0 or 10, depending on the absence or presence of stabilizing Mg2+ ions (0.01 M). 4. In the absence of divalent ions some aggregation of hemocyanin was found at pHs close to 5.0, with observed molecular weights above 10 x 10(6) (investigated at a hemocyanin concentration of 0.10 g/l). The early studies of Condie and Langer (Science 144, 1138-1140, 1964) had shown that Kelletia kelleti hemocynanin aggregates at acidic pHs close to the isoelectric point, forming linear polymers of the hemocyanin di-decamers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen binding by hemocyanin from Levantina hierosolima. I. Exclusion of subunit interactions as a basis for cooperativity.

The effect of oxygen on the distribution of hemocyanin from Levantina hierosolima among the three sedimenting species 20, 60, and 100 S was determined under two sets of experimental conditions: (a) at pH 7.63 in the absence of Ca2+, where oxygen binding in noncooperative; (b) at pH 8.20 in the presence of 2 x 10-3 M Ca2+, where oxygen binding is cooperative. A comparison of the results in the two cases eliminates the possibility that equilibrium between species with different oxygen affinities is responsible for the cooperative behavior. Cooperative oxygen binding was demonstrated for the 20S subunits at pH 8.80 and 1 x 10-3 M Ca2+. Under these conditions, the concentration of calcium is sufficient to affect the oxygen affinity, but the concentration of calcium plus proton is not sufficient to bring about association. The findings exclude interactions among 20S subunits as a basis for cooperativity in hemocyanin.     

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.     

Solubility and diffusion coefficient of adenosine 3':5'-monophosphate.

Several previously unavailable parameters of adenosine 3':5'-monophosphate have been determined. The molar extinction coefficient at pH 7.0 is 1.38 X 10(-4), the aqueous solubility at pH 7.0 is 0.0236 M and the diffusion coefficient is 4.44 X 10(-6) cm2/s.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

Effect of Ca++ on the structure and rheology of canine tracheal mucin

Mucin glycoproteins are known to be the principal determinants of epithelial mucus rheology and hence of mucociliary transport rates. We are studying the structure of such glycoproteins using a model mucin purified from canine tracheal pouch secretions. Of particular interest is the effect on mucin structure of increased Ca++ such as occurs in certain disease states. Quasielastic laser light scattering was used to study the effect of Ca++ on the hydrodynamic radius of the mucin molecules. Scattering data from 0.3mg/ml mucin solutions in physiological phosphate buffer containing 0, 5 X 10(-5)M, and 5 X 10(-4)M Ca++ were analyzed to obtain an average translational diffusion coefficient and the distribution of molecular radii for the dispersion. The effect of Ca++ was to decrease the average Stokes radius. The light scattering results are supported by rheologic measures of mucin gel viscoelasticity.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.     

Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis.

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5 X 10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.     

Light-scattering investigation of the subunit structure and dissociation of octopoda hemocyanins

The molecular weights, subunit dissociation, and conformation in solution of the hemocyanins of three species of octopi were investigated by light-scattering, ultracentrifugation, absorbance, and circular dichroism methods. The molecular weights of the hemocyanins of Octopus bimaculoides, Octopus bimaculatus, and Octopus rubescens obtained by light scattering were 3.3 X 10(6), 3.4 X 10(6), and 3.5 (+/- 0.3) X 10(6), respectively. The average molecular weights of the fully dissociated hemocyanins of the same octopi, investigated at alkaline pH and in the presence of 8 M urea and 6 M guanidinium chloride (GdmCl), were found to be close to one-tenth of those of the parent proteins, with average molecular masses of 3.4 X 10(5), 3.3 X 10(5), and 3.3 (+/- 0.3) X 10(5). These findings confirm the earlier observations of van Holde and co-workers with other cephalopod hemocyanins that the basic cylindrical assembly of molluscan hemocyanins consists of 10 subunits. Circular dichroism and absorbance measurements suggest that the dissociated subunits at alkaline pH and in concentrated urea solutions retain their native, multidomain folding. Fairly concentrated GdmCl above 3-4 M is necessary to unfold fully the dissociated hemocyanin chains. Molecular weight measurements studied as a function of reagent concentration with the urea and Hofmeister salt series as dissociating agents show that the ureas are very effective dissociating agents, while the salts are ineffective to moderately effective reagents for octopus hemocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent ion binding properties of the timothy grass allergen, Phl p 7

The timothy grass allergen, Phl p 7, was studied by calorimetry, spectroscopy, and analytical ultracentrifugation. As judged by isothermal titration calorimetry (ITC), the protein binds Ca (2+) cooperatively with stepwise macroscopic association constants of 1.73 x 10 (6) and 8.06 x 10 (6) M (-1). By contrast, Mg (2+) binding is sequential with apparent macroscopic association constants of 2.78 x 10 (4) and 170 M (-1). Circular dichroism and ANS fluorescence data suggest that Ca (2+) binding provokes a major conformational change that does not occur upon Mg (2+) binding. Conformational stability was assessed by differential scanning calorimetry (DSC). In phosphate-buffered saline (PBS) containing EDTA, the apoprotein undergoes two-state denaturation with a T m of 78.4 degrees C. In the presence of 0.02 mM Ca (2+), the T m exceeds 120 degrees C. Phl p 7 is known to crystallize as a domain-swapped dimer at low pH. However, analytical ultracentrifugation data indicate that the protein is monomeric in neutral solution at concentrations exceeding 1.0 mM, in both the apo and Ca (2+)-bound states.     

High affinity binding of divalent cation to actin monomer is much stronger than previously reported

Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca++ and Mg++ using the fluorescent Ca++ chelator Quin2 to induce and measure the dissociation of Ca++ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 +/- 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 X 10(6)M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca++ is 5.4 times that for Mg++, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought.     

Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogeneous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32,000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm epsilon = 2.34 X 10(4) M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 X 10(-8) M; JH II, 7.2 X 10(-8) M; JH III, 47 X 10(-8) M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, 11R)-JH II enantiomer. The pH optimum of binding is 7.0.