Effect of metallic cations and pH on reassembly of glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP), which was purified from acetone powder of the bovine spinal cord, was reassembled in 0.1 M imidazole HCl buffer containing metallic cations, Ca²⁺, Mg²⁺, Na⁺ or K⁺ at physiological or more acidic pH. An electron microscopy revealed reassembled glial filaments at pH 6.8 without any cations but amorphous aggregates at pH 6.3 which were readily observed as a white precipitate by the naked eye. Under more alkaline pH (pH 7.4) only rod-shaped short filaments were formed. In the presence of mM concentrations of Ca²⁺ or Mg²⁺, thick bundles of glial filaments, detectable by light microscopy, were formed at acidic pH. At pH 7.4 long reassembled filaments could be formed in the buffer containing divalent cations. Na⁺ (0.1 M) made filament-like structures of GFAP but they are rather random compared to the filaments promoted by the divalent cations. K⁺ made only amorphous aggregation of the short filaments. These findings indicate that the reassembly of GFAP at physiological pH requires essentially divalent cations but not ionic strength.     

In vitro reassembly of infectious polyoma virions.

Initial experiments in our laboratory have successfully reassembled infectious polyoma virions from dissociated virion products. Virions treated with ethyleneglycol-bis-N,N'-tetraacetic acid and the reducing agent beta-mercaptoethanol at pH 7.5 were dissociated to a 48S DNA-protein complex and capsomere subunits. The virion dissociation products were not infectious by plaque assay and lacked hemagglutination activity. These virion dissociation products were reassembled to intact virions by overnight dialysis against a reassembly buffer containing CaCl2, dimethyl sulfoxide, and Triton X-100 in phosphate-buffered saline at pH 7.4. The biophysical characteristics of the reassembled virions were identical to those of untreated virions in that the reassembled virions had a sedimentation value of 240S in sucrose gradients and a buoyant density of 1.315 g/cm3 in CsCl isopycnic gradients. The reassembled virions were intact as determined by electron microscopy and were found to be 60% resistant to DNase I treatment. Biologically, the reassembled purified virions were found to partially regain both hemagglutinating activity and plaque-forming ability.     

Subunit dissociation and denaturation of Fasciolaria tulipa hemocyanin

1. The hemocyanin from the marine snail, Fasciolaria tulipa has a molecular weight of 8.6 +/- 0.6 x 10(6) determined by light-scattering and a sedimentation constant of (105.9 +/- 1.1)S. 2. The dissociated subunits at pH 11 and in 8.0 M urea (pH 7.4) had molecular weights of 4.4 x 10(5) and 4.7 x 10(5), close to one-twentieth of the parent didecameric assembly. 3. The pH dependence of the molecular weight profile exhibited bell-shaped transitions in both the presence and absence of Ca2+ and Mg2+ ions. In the physiological pH range of about 7.5-8.2 in divalent ion-containing buffers neither the molecular weight behavior nor the sedimentation patterns suggest any significant dissociation. 4. Both the urea and the Hofmeister salt series were found to dissociate the didecameric hemocyanin assembly. The ureas exhibit increasing effectiveness as dissociating agents with the higher alkyl substituted members of the series, suggesting hydrophobic stabilization of the subunit assembly. 5. Denaturation of the hemocyanin subunits by the urea series follows the same trend in effectiveness as the dissociation reaction; the reagent concentrations required to cause unfolding of the globular domains of the hemocyanin chains were, however, much higher than those needed for 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.     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

Dissociation and oxygen equilibrium properties of earthworm (Pheretima hilgendorfi) hemoglobin

The dissociation and oxygen equilibrium properties of the purified hemoglobin and whole blood obtained from the earthworm Pheretima hilgendorfi were compared. Above pH 8.0, P1/2's were higher in the purified hemoglobin than in whole blood, while below pH 8.0, nearly identical P1/2's were observed in both materials and P1/2 was at a maximum at pH 6.5. The values of n1/2 were higher in whole blood than in the purified hemoglobin at alkaline pH. The maximum values of n1/2 were observed around pH 8.1 in the purified hemoglobin and pH 8.7 in whole blood, and the values were 5.3 and 9.5, respectively. In the purified hemoglobin, a small amount of dissociation component was already observed at pH 8.0, while in whole blood, no dissociation occurred up to pH 9.1. Dialysis of whole blood or addition of 10 mM EDTA to whole blood at alkaline pH induced the loss of the enhanced cooperativity, the increased oxygen affinity and the high stability of the 60 S whole molecule. These results strongly suggest that divalent cations are participating in the functional and dissociation properties of the whole blood of this species.     

Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Assembly of the fertilization membrane of the sea urchin: Isolation of a divalent cation-dependent intermediate and its crosslinking in vitro

To analyze the mechanism of assembly of the fertilization membrane of the sea urchin Strongylocentrotus purpuratus, we inhibited the ovoperoxidase that catalyzes dityrosine formation to isolate an uncrosslinked, soft fertilization membrane (SFM). The SFM intermediates were stabilized by divalent cation-dependent interactions: in the absence of divalent cations, the SFM became amorphous and less refractile and released proteins into the surrounding medium. We term the remaining structures “wraiths.” The rate of this disaggregation was increased in solutions of low ionic strength, but 510 mM divalent cations (Ca²⁺, Mg²⁺, Mn²⁺ or Ba²⁺) prevented disaggregation. Wraiths could be reassembled into structures that resembled SFM by readdition of divalent cations. The SFM contained active ovoperoxidase and could be hardened in vitro by washing away the ovoperoxidase inhibitor and adding H₂O₂. After hardening, certain proteins of over 100 kd were excluded from SDS-polyacrylamide gels, suggesting that these proteins contain the substrates for crosslinking. We propose that the SFM is a divalent cation-dependent intermediate on the pathway of fertilization membrane assembly containing tyrosyl residues that are appropriately juxtaposed for crosslinking.     

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)     

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L ‐lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI‐MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L ‐lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer‐specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L ‐lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effect of cations on the structure of mung bean cytoplasmic ribosomes

The importance of the absolute and relative concentrations of monovalent and divalent cations and centrifugal speed (pressure) in the dissociation of mung bean 80S ribosomes has been examined. In the absence of Mg²⁺ ions, ribosome monomers yield 47S and 34S particles. Fixation with glutaraldehyde, however, indicates that this dissociation pattern is largely dependent upon high pressures developed during centrifugation and that in the absence of such artifacts the immediate product of Mg-free conditions is a 74S particle. Since 74S particles rapidly revert to the 80S form when Mg is replaced, this would appear to be a conformational change. Ribosomes were also dissociated in the presence of Mg²⁺ ions if the K⁺ ion concentration was raised. Three major particles were produced, 38S and 49S from the small ribosomal sub-unit and 60S from the large sub-unit. A proportion of the 80S monomer population is more resistant to dissociation. Experiments with puromycin indicate that the more resistant fraction probably represents ribosomes completed with nascent polypeptide resulting from polysome breakdown.     

The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure

Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or "bracelet," decorated with 12 complexes of M and T subunits.     

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.     

The hemoglobin of the aquatic snail, Planorbella duryi (Wetherby)

1. The hemoglobin of the pond snail, Planorbella duryi has a molecular weight of 1.64 x 10(6) to 1.77 x 10(6) as determined by light-scattering at 630 nm and a sedimentation coefficient of 36 S. 2. The analysis of the circular dichroism spectrum obtained in the 190-250 nm region suggests a high degree of helical folding of the polypeptide chains of P. duryi hemoglobin analogous to human hemoglobin and myoglobin, with estimates of alpha-helical folding of about 60-65%, 0-5% beta-structure, and the remaining portion of the chains in unordered form. 3. The dissociated subunits in 6.0 M GdmCl, in the absence and in the presence of reducing reagent (0.1 M dithiothreitol), have a molecular weight of 3.73 +/- 0.23 x 10(5) and 1.93 +/- 0.04 x 10(5), suggesting a di-decameric assembly of the parent hemoglobin organized in the form of five dimers held together by disulfide-linkages. 4. The native hemoglobin is strongly resistant to both pH dissociation and dissociation by urea and such salts as NaCl and NaClO4. Dissociation and denaturation could only be effected in concentrated GdmCl solutions. 5. The influence of the various dissociating agents on the quaternary structure suggest ionic stabilization of the decameric assembly, which is stabilized by salt bridges between the subunits.     

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.     

A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from − 8 to − 10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.     

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.     

Dissociation and oxygen equilibrium properties of the extracellular hemoglobin of Eisenia foetida

The dissociation and oxygen equilibrium properties of whole blood and the purified hemoglobin from Eisenia foetida were compared. Oxygen affinities agreed approximately with each other in the range of pH 6.0 to 9.5. The values of n1/2 were higher in whole blood than in the purified hemoglobin between pH 7.0 and 9.5. The maximum values, obtained near pH 8, were about 6 in whole blood and 3.5 in the purified hemoglobin. In the purified hemoglobin, alkaline dissociation started at pH 7.8, and the approximately 60 S whole molecule dissociated completely into approximately 10 S and 5-6 S components at pH 9.1. In whole blood, however, the dissociation started at pH 8.2 and the complete disappearance of the approximately 60 S molecule occurred at pH 9.6. The values of n1/2 for the dissociation products were lower than those of the purified hemoglobin between pH 7.0 and 9.0. The value of n1/2 decreased with increasing dissociation of the approximately 60 S whole molecule with a pH rise in both whole blood and the purified hemoglobin. Addition of CaCl2 or MgCl2 up to 10 mM to the purified hemoglobin at pH 8.0-8.1 induced increases in oxygen affinity and cooperativity and in the stability of the approximately 60 S whole molecule. The effect on the oxygenation properties was greater with CaCl2 than MgCl2 at the same molar concentration. The stabilizing effect on the approximately 60 S molecule was almost the same with both CaCl2 and MgCl2. These results suggest that the dissociation of property of the hemoglobin in whole blood is controlled by both Ca2+ and Mg2+, and that its oxygenation property is controlled by Ca2+.     

Partial reassembly of yeast 60 S ribosomal subunits in vitro following controlled dissociation under nondenaturing conditions

Previously it has been shown that 12 of the yeast ribosomal proteins were extractable from 60 S subunits under a specific nondenaturing condition [J. C. Lee, R. Anderson, Y. C. Yeh, and P. Horowitz (1985) Arch. Biochem. Biophys. 237, 292-299]. In the present paper, we showed that these proteins could be reassembled with the corresponding protein-deficient core particles to form biologically active ribosomal subunits. Effects of time, temperature, and varying concentrations of monovalent cations, divalent cations, cores, and ribosomal proteins on reconstitution were examined. Reconstitution was determined by binding of radiolabeled proteins to the nonradiolabeled cores as well as activity for polypeptide synthesis in a cell-free protein-synthesizing system. The optimal conditions for reconstitution were established. Whereas the core particles were about 10-20% as active as native 60 S subunits in an in vitro yeast cell-free protein-synthesizing system, the reconstituted particles were 80% as active. The activity of the reconstituted particles was proportional to the amount of extracted proteins added to the reconstitution mixture. About 55 +/- 7% of the core particles recombined with the extracted proteins to form reconstituted particles. These reconstituted particles cosedimented with native 60 S subunits in glycerol gradients and contained all of the 12 extractable proteins.     

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.