Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1-7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.     

Molecular weight of Eudistylia vancouveri chlorocruorin and its subunits.

The chlorocruorin of the marine polychaete Eudistylia vancouveri has a molecular weight of 3.1-10(6) and a sedimentation coefficient (S020, w) of about 57 S at pH 8.0 in the presence of 0.01 M Mg2+. The quaternary structure of this pigment is unaffected by pH between 6.0 and 11.5 in the presence of 0.01 M Mg2+ whereas in 0l01 M EDTA, the pigment begins to dissociate above pH 9.0 into smaller submultiples. The chlorocruorin can be converted into subunits with molecular weights of about 14 000-15 000 and 30 000 as determined by sodium dodecyl sulfate-gel electrophoresis and 14 000-15 000 as measured by gel chromatography of the carboxy-methylated derivative in 8 M urea, 0.1 M 2-mercaptoethanol, or by sedimentation equilibrium in 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol. The pigment contains 0.212 +/- 0.008% iron corresponding to 1 g atom iron per 26 300 g chlorocruorin. The amino acid composition of this pigment is reported. The subunit structure of Eudistylia chlorocruorin and the polymeric annelid hemoglobins are similar in many respects.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established.     

Investigation of the subunit interactions in malate dehydrogenase.

The dissociations of porcine heart mitochondrial, bovine heart mitochondrial, and porcine heart cytoplasmic malate dehydrogenase dimers (L-malate: NAD+oxidoreductase, EC 1.1.1.37) have been examined by Sephadex G-100 gel filtration chromatography and sedimentation velocity ultracentrifugation. The porcine mitochondrial enzyme was found to chromatograph as subunits when applied to a gel filtration column at a concentration of .02 muM or less at pH 7.0. The presence of coenzymes shifted the dissociation equilibrium at low enzyme concentrations in favor of dimer formation. Monomer formation was also favored when procine mitochondrial enzyme was incubated at pH 5.0 even at concentrations as high as 120 muM. This shift in equilibrium has been correlated with the increased rate and specificity of sulfhydryl residue modification with N-ethylmaleimide at pH 5.0 (Gregory, E.M., Yost, F.J.,Jr., Rohrbach, M.S., and Harrison, J.H. (1971)J. Biol. Chem. 246, 5491-5497). Bovine mitochondrial enzyme did not exhibit a concentration-dependent disociation under the conditions examined. However, at pH5.0 monomer formation was favored, and correlations could again be drawn with sulfhydryl residue modification (Gregory, E.M. (1975)J.Biol. Chem. 250, 5470-5474). In both mitochondrial enzymes, coenzyme binding was found capable of overcoming the effects of pH on the dissociation equilibrium, and dimer formation was favored. Unlike either of the above mentioned enzymes, porcine cytoplasmic malate dehydrogenase did not dissociate into its monomeric form under any conditions investigated.     

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.     

Kinetics of deoxyhemoglobin subunit dissociation determined by haptoglobin binding: estimation of the equilibrium constant from forward and reverse rates.

Deoxyhemoglobin tetramers dissociate into dimers very slowly, with half-times on the order of several hours. It is demonstrated that absorbance changes in the Soret region which accompany this dissociation and persist upon binding of haptoglobin 1-1 to the dissociated dimers can be used for accurate kinetic determinations over the necessarily long periods required for study. This method of study for the slow reactions depends upon long-term spectral integrity of the reaction mixtures and upon accurate measurement. The variation in rate constants determined by this procedure has been correlated with variations in structural constraints at the dimer-dimer contact region. In the presence of 2,3-diphosphoglycerate the rate constant is decreased, consistent with the role of this effector in binding to both beta chains and stabilizing the constrained deoxy tetramer against dissociation into alphabeta dimers. With hemoglobin specifically modified (des-Arg-141alpha) to eliminate half the constraining salt links within the dimer-dimer contact region, the dissociation rate is increased by approximately three orders of magnitude. In hemoglobin S where the amino acid substitution is not directly in the intersubunit contact region of interest, the dissociation rate is found to be approximately the same as that for hemoglobin A. Combination of the dissociation rate constants determined by haptoglobin binding with stopped-flow determinations of the rate constant for reassociation of dissociated dimers provides an estimate of the equilibrium constant, 0K2, for the deoxyhemoglobin dimer-tetramer equilibrium. This estimate is independent of any assumptions regarding other energetic quantities, and yields a value of 2.54 +/- 0.7 X 10(10)M-1 (heme) in 0.1 M Tris-HCl, 0.1 M NaCl, and 1 mM EDTA, pH 7.4, 21.5 degrees C. Thus the intersubunit contact energy is -14.0 +/- 0.2 kcal/mol of heme. The stabilization energy between deoxy and oxy tetramers is found to be approximately 6.4 kcal/mol, under these conditions.     

Dissociation of human follicle-stimulating hormone. Comparison with luteinizing hormone.

Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.     

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...     

Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.

The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.     

Effect of Sulfhydryl Reagents on the Ribosomes of Bacillus subtilis

The effect of various sulfhydryl reagents on the ribosomes of Bacillus subtilis was studied. The 70S ribosomes were completely dissociated into 30S and 50S subunits by appropriate concentrations of p-chloromercuribenzoic acid (PCMB) and 5,5'-dithio-bis-(2-nitro-benzoic acid). The N-ethylmaleimide and iodoacetamide failed to dissociate the ribosomes even at relatively high concentrations. The rate of dissociation of ribosomes by PCMB varied with the concentration of ribosomes. A progressive decrease in the rate of dissociation was observed as the concentration of ribosomes in the reaction mixture was increased. The PCMB-induced ribosomal subunits were unable to reassociate into 70S monomers unless they were dialyzed against buffer containing beta-mercaptoethanol. On the average, four molecules of PCMB per 70S ribosome and two molecules of PCMB per each 30S and 50S subunit were bound. The number of PCMB molecules bound per ribosome did not change with increasing concentrations of PCMB, even though higher concentrations of PCMB resulted in dissociation of ribosomes into subunits.     

Dissociation of eukaryotic ribosomes by purified initiation factor EIF-3

Purified eukaryotic initiation factor, EIF-3, prepared from ascites cells dissociated rat liver 80S ribosomes into subunits. Ribosomes bearing endogenous mRNA and nascent peptide were not dissociated by EIF-3. When 80S ribosomes reconstituted from subunits were used as substrate the reaction had the following characteristics: Dissociation was rapid--the reaction being completed within 2 min at 30°. The extent of dissociation was directly proportional to the amount of EIF-3; with 21 μg of EIF-3 about 70% (or 10.5 μg) of the 80S monomers were dissociated. The dissociation of 80S monomers by EIF-3 decreased with increasing concentrations of magnesium. The reaction was not catalytic: 28 moles of EIF-3 were required to dissociate 1 mole of 80S ribosomes. The characteristic of the dissociation reaction promoted by EIF-3 and by $\text{E. coli}$ initiation factor IF-3 are remarkably similar. The dissociation reaction provides a practical assay for EIF-3 independent of complimentation of other initiating factors.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

The effect of alkaline earth cations and of ionic strength on the dissociation of earthworm hemoglobin at alkaline pH

1. The effect of alkaline earth cations on the dissociation of the extracellular hemoglobin of Lumbricus terrestris and the effect of ionic strength on the dissociation of the hemoglobins of L. terrestris and Tubifex tubifex at concentrations of ca 2.5 mg/ml, over the pH range 9.0-10.5 was investigated using ultracentrifugation to separate the dissociated from the undissociated molecules. 2. Mg(II), Ca(II) and Sr(II) at concentrations of up to 0.2 M, decreased the dissociation of Lumbricus oxyhemoglobin from 70% at pH 9.0 and 100% at pH 9.5 and higher, to 20-30% at 0.05 M. The three cations were equally effective in decreasing the extent of dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5, with a K1/2 of ca 10 mM. 3. The dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5 was decreased only to 50-60% in the presence of up to 0.5 M NaCl or KCl; there was no further decrease in dissociation at concentrations of the two salts up to 1.5 M. 4. The dissociation of T. tubifex oxyhemoglobin over the pH range 9.0-10.0 was decreased from 100% to ca 40-50% in the presence of 0.5 M NaCl or KCl with little or no change at higher concentrations. At pH 10.5 and 11.0 the decrease in dissociation was more gradual, reaching ca 50% at 1.5 M NaCl.     

Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Porcine spleen DNase II (EC 3.1.22.1), one of the best-characterized DNases II, is subcellularly located in lysosomes because the enzyme is co-sedimented with two of the lysosomal marker enzymes, cathepsin D and acid phosphatase. The physicochemical properties, including the subunit structure, sensitivity to iodoacetate inactivation, native molecular weight and chromatographic behavior, of the DNase II purified from the isolated lysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase II can also be extracted from porcine liver with 0.05 M H2SO4 or 0.1 M NaCl and purified from either extract by a series of column chromatographies. The purified liver DNase II from either extract has the same subunit structure (alpha-chain, Mr 35,000 and beta-chain, Mr 10,000) as the purified DNase II of porcine spleen. The two liver extracts as well as the extracts of spleen and gastric mucosa contain DNase II with very similar properties on Sephadex G-100 gel filtration, on acid polyacrylamide gel electrophoresis under non-denaturing conditions, and on isoelectric focusing. The data strongly suggest that, for the same species of animal, the DNase II activities in various tissues are associated with protein molecules of identical structure.     

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.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Rates of dissociation and recombination of the subunits of bovine thyrotropin

The rates of dissociation and recombination of the subunits of bovine thyrotropin have been measured under a variety of conditions using the fluorescence probe 1,8-anilinonaphthalenesulfonate. The method is based on the fact that the native hormone strongly enhances the fluorescence of 1,8-anilinonaphthalenesulfonate whereas the subunits have very little effect. The hormone can be easily dissociated into subunits, either in dilute acid (pH < 4) or in concentrated (8–10 m) urea solutions at pH 8.O. The rate of dissociation is first order with time and increases strongly with increasing temperature. The hormone is very stable in alkali, showing little tendency to dissociate below pH 12. After dissociation in acid, the subunits can be recombined between pH 7 and 9 at a rate which increases with increasing temperature and subunit concentration. The recombination is intermediate between first and second order suggesting a two-step mechanism: association of the subunits followed by a first-order refolding process in which the subunits acquire the tertiary structure characterisitc of the native hormone. Difference absorption measurements indicate that the dissociation is accompanied by the exposure of a substantial fraction of the 16 tyrosine residues to the more polar aqueous environment, suggesting major conformational changes in one or both subunits.     

Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8-10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5-5.0, the subunit reassociates to oligomer in the pH range 3.1-4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.     

The hemocyanin of the ramshorn snail, Marisa cornuarietis (Linné)

1. The hemocyanin of the freshwater snail, Marisa cornuarietis exists predominantly as a di-decamer with the approximate mol. wt of 8.5 x 10(6) and a sedimentation coefficient of 100 S. Sedimentation and scanning transmission electron microscopy experiments indicate that about 15-20% of the hemocyanin forms tri-decameric and possibly higher aggregates with mol. wts of 12.5 x 10(6) and 130 S. 2. The fully dissociated subunits in 8.0 M urea and 6.0 M GdmCl have mol. wts of 4.1 to 4.7 x 10(5) which is close to one-twentieth of the major di-decameric component of the native hemocyanin. 3. Subunit dissociation by the urea series and the Hofmeister salt series of reagents suggests hydrophobic stabilization of the decamers or half-molecules of the parent hemocyanin. As with the other molluscan hemocyanins the order of effectiveness of the ureas as dissociating agents shows increased efficacy with increasing hydrophobicity or chain-length of the urea substituents. 4. Denaturation of the hemocyanin subunits by the ureas and Hofmeister salt series, investigated by circular dichroism measurements, essentially follow the same trend in effectiveness as observed by changes in subunit dissociation followed by light-scattering mol. wt measurements. 5. The observed denaturation transitions are shifted to much higher ranges of reagent concentration than the concentrations required for the dissociation of the hemocyanin subunits.