PROPERTIES OF THE PROTEIN SUBUNIT OF CENTRAL-PAIR AND OUTER-DOUBLET MICROTUBULES OF SEA URCHIN FLAGELLA

The subunit protein has been isolated from the central-pair and outer-doublet microtubules of sea urchin sperm tails. Both proteins have a sedimentation constant of 6S and a molecular weight of 120,000. Both are converted to a 60,000 molecular weight species by denaturation in 6 M guanidine hydrochloride and reduction with mercaptoethanol. The reduced-alkylated proteins have the same R_f on disc electrophoresis, and the same amino acid composition, which is very similar to that of muscle actin. The central-pair protein has one binding site for colchicine per 120,000 g. Both proteins appear to have a guanine nucleotide binding site, but the ability to bind GTP in solution has been demonstrated only for the central-pair protein. Although 1 mole of guanine nucleotide is bound per 60,000 g to outer-doublet tubules, the protein obtained by dissolving the doublets at pH 10.5 has lost the guanine nucleotide-binding site and also shows little or no colchicine-binding activity. Comparison of the properties of the isolated protein with electron microscopic evidence on structure of microtubules suggests that the chemical subunit (M = 120,000) consists of two of the 40 A morphological subunits.     

The subunit structure of Pseudomonas cytochrome oxidase.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.     

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.     

Dissociation of ferritins

Apoferritins prepared from horse spleen and heart and rat heart and liver were dissociated by treatment with acetic acid (pH 1.3-3.0). Sedimentation velocity studies showed that apoferritins of spleen and liver (16-17 S) and heart (18-19 S) dissociated into material sedimenting near 3.2 S. Sedimentation equilibrium measurements determined that most of the material had a molecular weight of 38,000-43,000, corresponding to subunit dimers. Failure to dissociate into subunit monomers was confirmed by gel chromatography on Sephadex G-75 and G-150. With the exception of boiling in sodium dodecyl sulfate, further treatments with 0.1-0.4 M KCl, NaCl, 4-9 M urea, 0.01-0.5 M KSCN, 0.1-0.5% Triton X-100, 5-52% dimethylsulfoxide, 10% ethylene glycol, or 0.1% trifluoroacetic acid all failed to cause dissociation into individual subunits, as did exposure to 6 M guanidine-HCl or formic acid, or prior succinylation and/or nitration of the protein. Reassociation occurred between pH 4 and 7 but was not aided by the addition of Fe(II) or reducing agents. It is concluded that ferritins readily dissociate to subunit dimer units and that further dissociation does not occur without full denaturation of the protein.     

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.     

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.     

Rat Brain α-Mannosidase: Purification, Properties, and Interaction with Its Antibodies

Abstract: α - d -Mannosidase (EC 3.2.1.24.) was purified to homogeneity from adult rat brain. The enzyme, of apparent molecular weight 397,000, appears to be formed of subunits of molecular weight 120,000 made of two protomers (62,000) bound by disulfide bridges. Isoelectric focusing gives two bands, of pi 5.40 and 5.15. Both isoenzymes seem to have the same pH curve (a small peak of activity at pH 4.5 and a maximum of activity around pH 6.0). These two isoenzymes are immunologically related.     

Enzymatic methyl esterification of Escherichia coli ribosomal proteins.

Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

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.     

Purification and structural analysis of a latent form of transforming growth factor-beta from rat platelets

A latent form of transforming growth factor type-beta (TGF-beta) with a high molecular weight was purified to homogeneity from rat platelets by a six-step procedure. The yield of the purified latent TGF-beta from platelets of 2,500 rats was 1.4 mg. The purified latent TGF-beta was activated by treatment with urea at concentrations of over 4M or acidic solutions of below pH 4. SDS-PAGE and gel filtration chromatography showed that the latent TGF-beta consisted of active TGF-beta and glycoproteins of about 200 kDa as masking components, and that under physiological conditions, these components formed a high molecular weight complex of about 400 kDa linked by non-covalent bonds. Here, we found that the masking protein was composed of one large subunit of about 110 kDa and two small subunits of 39 kDa linked by disulfide bridges. The N-terminal amino acid sequence of the small subunit was identical to the N-terminal region of the TGF-beta precursor lacking a signal peptide. From these findings, we proposed a structural model for the latent TGF-beta from rat platelets.     

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)     

Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.

The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.     

Bacterial membrane proteins. Properties of Micrococcus lysodeikticus NADH dehydrogenase]

NADH dehydrogenase was isolated from M. lysodeikticus membranes with FAD as a prosthetic group. It was found the enzyme molecular weight is about 140000 in 0,01 M phosphate buffer, pH 7,4 in 1% Triton X-100. The enzyme molecules are dimers consisting of two subunits with molecular weight of 70000. The content of alpha-helical regions is 30%, that of beta-forms is 13%. The protein globule is cross-linked with the disulfide bonds and has hydrophobic regions on its surface.     

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)     

Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.     

Tryptophanase from Sphaerophorus funduliformis. Purification, molecular weight and subunit properties.

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH4)2SO4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5-13.5 and requires pyridoxal 5'-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine - HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.     

Structural characterization of insulin receptors. II. Subunit composition of receptors from turkey erythrocytes

In the preceding paper (Aiyer, R. A. (1983) J. Biol. Chem. 258, 14992-14999), the hydrodynamic properties of insulin receptors from turkey erythrocyte plasma membranes solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate) were characterized. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peak II whose protein molecular weight (Mp) is 180,000 +/- 45,000 and its disulfide-linked dimer, peak I (Mp, 355,000 +/- 65,000). This paper describes the subunit composition of these species determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Insulin receptors were covalently attached to [125I]iodoinsulin with disuccinimidyl suberate. After solubilization in Triton X-100 or deoxycholate, peaks I and II were separated by sedimentation and subjected to SDS-PAGE; the constituent polypeptides were then identified by autoradiography. Under reducing conditions, both peaks I and II yield a major band of apparent molecular weight (Mapp) of 135,000; this band most likely represents the insulin-binding subunit (alpha). Minor bands of lower molecular weight are also seen whose significance is not entirely obvious. Under nonreducing conditions, peak I yields bands at Mapp = 230,000 and at greater than 240,000, while peak II yields bands at Mapp = 120,000 and 200,000. When these bands were cut out of the gel and subjected to SDS-PAGE following reduction with 10% beta-mercaptoethanol, all of them produced a single band that migrated with Mapp = 135,000. These results indicate that the alpha subunit is linked by disulfide bonds to at least one more subunit (beta). It is also apparent that the alpha subunit travels with higher mobility (Mapp = 120,000) under nonreducing conditions, suggesting the presence of intrachain disulfide bonds. Thus, peak II has a minimum subunit composition of alpha beta, where alpha is the insulin-binding subunit with a minimum Mr = 120,000-135,000 and beta has a minimum Mr = 80,000-90,000. And peak I, the disulfide-linked dimer of peak II, has a minimum subunit composition of alpha 2 beta 2. These results were further confirmed by cross-linking of protein subunits with glutaraldehyde, an (alpha, omega)-dialdehyde that reacts with amino groups. Within the limits of error, these molecular weights are in agreement with those estimated from the hydrodynamic properties of the detergent-solubilized, native receptor species reported in the preceding paper.     

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)