Dissociation and denaturation behaviour of sesame alpha-globulin in sodium dodecyl sulphate solution.

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, alpha-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of .175-4.0 X 10(-"3) M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 x 10 (-3) M detergent suggesting conformational change; above 8 X 10(-"3) M detergent the value of -[alpha] does not change. The reduced viscosity etared however, increases above .25 X 10(-3) M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an "isoemissive point" at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5-0 X 10(-3) M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and binding constant of 3-0 X 10(3).     

Dissociation, aggregation of sesame alpha-globulin in nonionic detergent solution.

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame alpha-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pKInt value of the tyrosyl groups at 1x10-3 M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

The effect of prostaglandins E1, E2, F1 alpha and F2 alpha on pig erythrocytes during haemolysis induced with aspirin and hypotonic NaCl solution

The effect of prostaglandins PGE1, PGE2, PGF1 alpha and PGF2 alpha was investigated on the haemolysis of pig erythrocytes induced with aspirin and hypotonic (0.119 M) NaCl solution. An inhibiting effect was observed of low concentrations (2 X 10(-5) M, 2 X 10(-4) M and 2 X 10(-3) M) of aspirin on haemolysis induced with hypotonic NaCl solution, while in a concentration of 2 X 10(-2) M aspirin itself caused haemolysis which amounted to 93% of the haemolysis induced with 0.041 M NaCl solution. No differences were observed in the degree of haemolysis inhibition in relation to the time of incubation of erythrocytes with aspirin. Aspirin concentrations from 0.035 M to 0.280 M caused slight haemolysis (9-15% of the haemolysis induced with water), the 0.560 M solution caused haemolysis corresponding to 85% of the water-induced haemolysis. None of the studied prostaglandins used in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M had any significant effect on aspirin-induced haemolysis. PGE1 and PGE2 in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M inhibited haemolysis induced with 0.119 M sodium chloride solution, and the degree of haemolysis inhibition was from 8% to 35%. Prostaglandins PGF1 alpha and PGF2 alpha in the same concentrations had no protective effect.     

Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C

Monoclonal antibodies to various domains of human protein C were characterized, and the cross-reactivity of these antibodies with other vitamin K-dependent proteins was explored. Three antibodies, JTC-1, -2, and -3 reacted with protein C only in the presence of Ca2+ and were shown to bind to the light chain of protein C. It is suggested that these antibodies recognize a gamma-carboxyglutamic acid domain-related conformational change induced by metal ions, evidenced by the fact that half-maximal binding was observed at calcium concentration of 0.5, 0.6, and 0.7 mM, respectively, by the fact that these antibodies, even in the presence of Ca2+, do not react with gamma-carboxyglutamic acid domainless protein C, and by the fact that Zn2+ and Tb3+ support binding in essentially the same way. Each cell line was stabilized by recloning five times. In addition each antibody had a single isoelectric point and was of the IgG1 kappa class. The interaction of antibodies JTC-1, -2; and -3 with protein C-Ca2+ was characterized by a single class of binding sites with Kd of 3.98 X 10(-9) M, 4.01 X 10(-9) M, and 6.76 X 10(-9) M, respectively. However, antibodies JTC-1, -2, and -3 bound to prothrombin-Ca2+ with Kd of 7.81 X 10(-9) M, 2.0 X 10(-7) M, and higher than 1.0 X 10(-5) M, respectively. In addition they had weak affinity for factor X in the presence of Ca2+. The results indicate that the antibodies JTC-1, -2, and -3 are conformation-specific monoclonal antibodies directed against an at least partially common metal ion-induced three-dimensional structure in protein C, prothrombin, and factor X.     

Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro

The inadequate insulin secretory response to glucose stimulation in non-insulin dependent diabetes has been attributed to many factors including high PGE2 levels blunting the secretory response, and to the existence of inhibitory opiate activity in vivo. The purpose of the present work was to see if there was a connection between these two independent theories. Radioimmunoassayable PGE2 in islets of Langerhans was found to be proportional to islet number and protein content and was typically 4 to 5pg/micrograms islet protein. Indomethacin (2.8 X 10(-5) M), sodium salicylate (1.25 X 10(-3) M) and chlorpropamide (7.2 X 10(-5) M) all lowered islet PGE2 levels and stimulated insulin release in vitro. Dynorphin (1-13), stimulated insulin release at a concentration of 6 X 10(-9) M, while lowering islet PGE2. Conversely, at a higher concentration, (6 X 10(-7) M), dynorphin had no stimulatory effect on insulin secretion and did not lower PGE2 levels in islets or in the incubation media. The stimulatory effects of dynorphin and sodium salicylate on insulin secretion were blocked by exogenous PGE2 (10(-5) M). PGE2 at a lower concentration (10(-9) M) did not exert any inhibitory effect on dynorphin- or sodium salicylate-induced insulin release. This concentration of exogenous PGE2 stimulated insulin release in the presence of 6mM glucose. Results from these experiments suggest that since an opioid peptide can lower endogenous PGE2 production in islets and since the stimulatory effects of the opioid peptide are reversed by exogenous PGE2 there may be interactions between these two modulators of insulin secretion.     

Effect of ethacrynic acid on contractility and Ca flux of guinea pig taenia coli smooth muscle (author's transl)

The effects of ethacrynic acid (ETCA) which has been known as an -SH groups inhibitor on the contractility and the Ca flux of guinea pig taenia coli were investigated. The results obtained were as follow: 1) Contractures induced by 10(-4) M ACh, or the tonic component of 150 mM K-contractures were markedly suppressed by previous treatment with a low concentration (2 X 10(-4) M) of ETCA for 40 min. Conversely with the same treatment, the phasic component of this K-contracture was only slightly suppressed. The inhibitory effects of ETCA in both cases were reversed by the repetitive washing out of ETCA from taenia coli with normal tris-buffered solution. 2) ETCA, at concentrations higher than 10(-3) M, more markedly inhibited the ACh-, and the K-contractures. In this case these inhibitions were irreversible. 3) Cysteine in an equimolar concentration of ETCA prevented the inhibitory effects of ETCA on both contractures. 4) ETCA (10(-4) M) inhibited the ACh-contracture in Ca2+-free isotonic KCl solution to approximately the same degree as that in normal solution. 5) Inhibition of ACh-contracture by ETCA in Na+-free isotonic LiCl solution was less than that in normal solution. 6) ETCA (2 X 10(-4), or 10(-3) M) markedly stimulated 45Ca efflux from taenia coli in 20 mM Ca-EGTA tris-buffered solution. 7) 45Ca efflux acceleration by ETCA in Na+-free (replaced by Li+) 20 mM Ca-EGTA tris-buffered solution was less than that in 20 mM Ca-EGTA tris-buffered solution. These results may be explained by assuming that the inhibitory effect of ETCA on ACh-contracture can be attributed to the depletion of stored intracellular Ca and the acceleration of Ca efflux as a result of ETCA treatment.     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

Histone subunit interactions as investigated by high pressure

High-pressure fluorescence polarization was used to investigate subunit interactions of the histone H2A-H2B dimer and the H3/H4 tetramer isolated from calf thymus (CT) and chicken erythrocyte (CE) chromatin. The proteins were individually labeled with the fluorescent probe 5-(dimethylamino)-naphthalene-1-sulfonate (dansyl or DNS), and the fluorescence polarization was measured as a function of pressure. The long fluorescence lifetime of the probe allows for the observation of global rotations of the protein, the rate of which is dependent upon the aggregation state. From the pressure dependence of the dansyl polarization, the Kd of H2A-H2B dissociation of the CE dimer was found to be approximately 1 X 10(-7) M at 2.0 M NaCl. Lowering the salt concentration to 200 mM slightly stabilized the protein to 6 X 10(-8) M. Our data indicate a small negative volume change for the dissociation of the core particle octamer. The (H3)2(H4)2 tetramer, as was shown in the previous paper (Royer et al., 1989), also formed predominantly dimers of tetramers at higher protein or salt concentrations. In the study presented here, we found the dissociation constant for the H3/H4 octamer to dimer transition to be 1 X 10(-21) M3 (C1/2 = 4 X 10(-8) M) at 2 M NaCl for the CT preparation. Decreasing the salt concentration to 200 mM reduced the stability of the CT H3/H4 octamer to 9 X 10(-21) M3 (C1/2 = 8 X 10(-8) M). The dimer of the CE tetramer also dissociated upon application of pressure in 2 M salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes

Bovine thyroid membranes are able to dephosphorylate exogenous [1-3H]DMP as well as endogenous prelabeled [32P]DMP. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1-3H]dolichol from [1-3H]DMP and the residual amount of [32P]DMP. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. Triton X-100 is necessary for enzymatic activity with [1-3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [32P]DMP. Divalent cations are not essential for enzymatic activity, Ca2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO4(3-) are strongly inhibitory. The inhibition by dolichol and PO3-4 can be explained as the result of product inhibition. An apparent Km-value of 2.5 X 10(-5) M is computed for [1-3H]DMP. Bacitracin inhibits DMP-phosphatase in contrast with other reports. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting

The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.     

The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.     

Ca2+-surfactant interactions affect enzyme stability in detergent solutions

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.     

The thermal depolymerization of porcine submaxillary mucin

The time dependence of the molecular weight, radius of gyration, and hydrodynamic size distribution for porcine submaxillary mucin (PSM) in solution have been studied using static and dynamic light scattering. The weight average molecular weight (Mw) of PSM in 6 M guanidine HCl, pH 7, is initially 3 X 10(6) and decreases with time in three phases: rapidly from 3-2 X 10(6), less rapidly from 2-0.9 X 10(6), and slowly below 0.9 X 10(6). The rates of decrease are much greater at pH 2. The energy of activation associated with each phase is 20 kcal/mol, which is similar to that reported for peptide bond cleavage at an aspartic acid residue. Addition of mercaptoethanol to PSM in 6 M guanidine HCl leads to a rapid decrease in Mw to 0.9 X 10(6), followed by a very slow further decrease. These results suggest that native PSM consists of subunits (Mw = 0.9 X 10(6] that are linked by disulfide bonds to form dimers (Mw = 2 X 10(6] and then higher aggregates. This cross-linking appears to occur at unglycosylated regions of the protein core, which are believed to be richer in aspartic acid than the rest of the molecule.     

Optical and EPR spectroscopy studies on haem arginate, a new compound used for treatment of porphyria

A protohaem compound, used for treatment of porphyrias, has been studied to elucidate its state of aggregation. EPR and absorption spectroscopy measurements reveal that 38.3 mM protohaem, dissolved in 40% 1,2-propanediol/10% ethanol/water solution, also containing 153 mM arginine, is partly EPR silent. It exists as high molecular weight aggregates and probably also as mu-oxo-dimers. Dilution in the aqueous alcohol solution dissolves the aggregates first to oligomers and dimers, and finally to monomers (Kdiss = 24 X 10(-6)M). When haem is diluted in 0.9% sodium chloride, a fully monomeric state is not reached even at 1 microM concentration. At 3.5 microM concentration, that used for infusion in patients, the haem is still totally aggregated.     

Self-association of myelin basic protein: enhancement by detergents and lipids

Self-association of basic protein has been proposed to be of functional significance in central nervous system myelin. In aqueous solution this protein self-associates, previous data being consistent with the formation of dimers, which then undergo an indefinite isodesmic self-association [Smith, R. (1980) Biochemistry 19, 1826-1831]. As this protein is membrane bound in vivo, we have now examined the effects of amphiphiles on the self-association equilibria. Contrary to the expected effects, at low molar ratios dodecyl sulfate, deoxycholate, Triton X-100, and lysophosphatidylcholine increased protein intermolecular attraction. The anionic detergents led to partial precipitation even at 1:1 protein:detergent molar ratios whereas the zwitterionic lipid and the nonionic detergent exerted less pronounced effects. Sedimentation velocity and equilibrium measurements have been used to define quantitatively the effects of lysophosphatidylcholine. The sedimentation coefficient increases up to a lipid:protein ratio of 4:1 and then remains constant up to a ratio of 12:1. The sedimentation equilibrium data suggest that the mode of protein-protein interaction is the same as in the absence of lipid but with substantially increased association constants. The dimerization constant is increased from 1.20 X 10(2) M-1 to 1.0 X 10(3) M-1 and the isodesmic association constant from 3.4 X 10(4) M-1 to 1.2 X 10(5) M-1. The effects of detergents on myelin basic protein are compared with the effects on other proteins, and the implications for the state of the protein with myelin are discussed.     

Self-association of the major protein component of bovine serum high density lipoprotein.

The major protein component of bovine high density lipoprotein was investigated in solution by fluorescence polarization and ultracentrifugal techniques. A fluorescent derivative of this protein with 1-dimethylaminonaphthalene-5-sulfonyl chloride was employed in the fluorescence experiments. Over the concentration range from 5-10(-7) M to 5-10(-4) M of the protein monomer at pH values from 2 to 11 and ionic strengths from 0.03 to 2.0, at 23 degrees C, the major protein of bovine high density lipoproteinapoprotein (Apo-HOL-I) was found to exist in a stable aggregated form. The aggregate was not affected by dioxane additions of up to 20% nor by Triton X-100 to 0.2%, but dissociated readily in the presence of 0.07% sodium dodecylsulfate or 6 M urea. At concentrations below 5-10(-7) M, dissociation of the protein aggregate started spontaneously and continued down to 10(-8) M, the lowest measurable concentration. Several physiocochemical properties of the major protein of bovine high density lipoprotein were determined in the stable aggregate form. Molecular weight was 104 000 from ultracentrifugal analysis and 80 000 from gel-filtration. Rotational relaxation time was 115 ns at 25 degrees C, and s-0 20,w was 4.78 s. The results suggest very strong protein-protein interactions (Kd less than 10(-7) M) that are not electrostatic in nature. Hydrophobic interactions of a magnitude that could be affected by 20% dioxane or 0.2% Triton X-100 detergent are also excluded. There is saturation of the interaction sites by the aggregation of a few protein monomer units possibly to form a tetramer which is moderately asymmetric (1:4 axial ratio, assuming an ellipsoid of revolution) and relatively rigid. The strong protein-protein interactions in this pure apolipoprotein suggest the possibility of competition of inter-protein associations with protein-lipid interactions in in vitro lipid binding or lipoprotein reconstitution experiments.