Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Cross reaction of antibodies against liver gap junction protein (26K) with lens fiber junction protein (MIP) suggests structural homology between these tissue specific gene products

Detergent solubilization of γ-glutamyl transpeptidase was deduced from measuring sedimentation equilibrium behaviour in an air-driven ultra-centrifuge. Sequential fractionation of the tube contents and analysis of the radial concentration dependence permits direct determination of molecular weight. Studies using D₂O and H₂O indicate that the Triton X-100 solubilized enzyme has a molecular weight of approximately 175,000 and contains 55% bound detergent. The glycoprotein nature of the enzyme is demonstrated by its interaction with concanavalin A.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

Physical properties of the purified cardiac muscarinic acetylcholine receptor

The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoreaction center of photosynthetic bacteria. 2. Size and quaternary structure of the photoreaction centers from Rhodospirillum rubrum strain G9 and from Rhodopseudomonas sphaeroides strain 2.4.1.

The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha.     

Hydrodynamic properties of rat hepatic prolactin receptors

The hydrodynamic properties of rat hepatic prolactin receptors have been determined by a combination of gel chromatography and ultracentrifugation. Prolactin receptors were detergent extracted from partially purified plasma membranes prepared from female rat livers. Fifteen different nonionic detergents were tested for solubilizing prolactin receptors, including Triton X-100, Polyoxyethylene W-1, Lubrol WX, detergents of the Tween and Brij series, and digitonin. When the receptors were detergent solubilized after ligand was bound to the receptor, 1% Triton X-100 had the highest efficacy of solubilization. However, if the receptors were solubilized prior to exposure to ligand, maximum binding was to receptors solubilized with 0.25% Triton X-100. The K_d of 43.2–74.5 pM for binding to the soluble receptor was three to fivefold lower than the K_d for the membrane receptor. Gel chromatography (Bio-Gel A-1.5m, 2.5 × 50 cm) of the soluble receptor indicated a Stokes radius (R_s) of 5.0 nm for the hormonereceptor-detergent complex. The hydrodynamic properties of the receptor-detergentligand complex were determined by centrifugation in 5–20% sucrose gradients in H₂O and in D₂O. They are $\text{v}\text{?}\text{= 0.7; s}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}\text{= 4.7;}\text{f}\text{f}{}_0\text{= 1.49; M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 118,000}$ for the complex, 73,000 for the receptor alone. Approximately 0.22 mg of Triton X-100 is estimated bound per milligram of protein. This represents about 25 mol detergent/mol receptor.     

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H⁺,K⁺-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H⁺,K⁺-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C₁₂E₈, followed by exchange of C₁₂E₈ with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s_20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H⁺,K⁺-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α₂,β₂ dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H⁺,K⁺-ATPase. Thus, α,β H⁺,K⁺-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca²⁺-ATPase and Na⁺,K⁺-ATPase.     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Differential interferometry in the analytical ultracentrifuge. II. General applications

Various ways of applying differential interferometry to ultracentrifugal analyses are examined and several analytical techniques are established. In transport and moving boundary methods, the sedimentation coefficient is more precisely determined in the differential interference system than in the schlieren optical system because fringe measurement accuracy is much higher in the former system. Compared to interference and absorption optics, the differential interferometer provides a more exact s value in the transport method since an accurate calculation procedure can be adopted. Moreover, the following advantages of differential interferometry are noted. Determination of the initial solute concentration, which must be done in the usual interference method, is unnecessary in this sedimentation equilibrium method. Regardless of the partial loss of solute from the observed system due to rapid precipitation or adsorption to the cell wall during centrifugation, the molecular weight of the rest of the solute can be determined exactly. The diffusion coefficient can be determined accurately by fringe displacement analysis at the hinge point during the transient state. Together with the molecular weight and diffusion coefficient, the partial specific volume and sedimentation coefficient of a solute can be obtained from the result of a single low-speed centrifugation when the sample solutions in H₂O and D₂O are compared.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Structural characterization of insulin receptors. I. Hydrodynamic properties of receptors from turkey erythrocytes

Insulin receptors from turkey erythrocyte plasma membranes were solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate). Their hydrodynamic properties were determined by sedimentation analyses in H2O and D2O, and gel filtration on Sepharose 4B. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peaks I and II. In Triton X-100, the sedimentation coefficient (s20,w), partial specific volume (Vc), and Stokes radius (a) for peaks I and II are, respectively, 10.2 +/- 0.5 S and 6.6 +/- 0.5 S, 0.75 +/- 0.02 ml/g, and 0.76 +/- 0.02 ml/g, and 89 +/- 3 A and 76 +/- 3 A, to yield Mr = 410,000 +/- 75,000 and 235,000 +/- 55,000, respectively, for the protein-Triton X-100 complex. The corresponding values in deoxycholate solution are: 10.7 +/- 0.5 S and 6.9 +/- 0.5 S, 0.71 +/- 0.03 ml/g and 0.70 +/- 0.04 ml/g, and 86 +/- 3 A and 69 +/- 3 A for peaks I and II, respectively, to yield 360,000 +/- 65,000 and 180,000 +/- 45,000, respectively, for the molecular weight of the protein-deoxycholate complex. These data are consistent with a model whereby each receptor species binds to one micelle of the appropriate detergent. In agreement with this model, it was also found that, in both Triton X-100 and deoxycholate, concentrations higher than the critical micellar concentration are required in order to maintain discrete receptor species in solution. At concentrations below the critical micellar concentration, the receptors aggregate to a broad band that sediments faster than 11.3 S. This is typical of membrane proteins that are stabilized in solution by insertion into detergent micelles. Based on these results, the protein molecular weights of peaks I and II are estimated to be 355,000 +/- 65,000 and 180,000 +/- 45,000, respectively. When membranes are treated with the reducing agent dithiothreitol, peak I is converted to peak II. This fact, together with the estimates obtained for the protein molecular weights of the two receptor species, suggests that peak I is a disulfide-linked dimer of peak II. The sedimentation characteristics of insulin receptors in many different cell types appear to be similar. As with turkey erythrocytes, detergent extracts of membranes from rat liver contained two native receptor species whose sedimentation coefficients were similar to peaks I and II. However, in all the other cell types examined, including rat adipocytes, rat heart muscle, 3T3-L1 adipocytes, 3T3-C2 fibroblasts, and FAO hepatoma cells, peak I (the native dimer) was the predominant species observed.     

Dimeric nature of apo-low density lipoprotein extracted with Triton X-100.

Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.     

The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91,000 (glycosylated) and 22,000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b/detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3-1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the Mr = 91,000 and Mr = 22,000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has Mr values of 110,000-120,000 and 120,000-135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the Mr = 91,000 and Mr = 22,000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carrying subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an alpha-beta-type heterodimer composed of a glycosylated Mr = 91,000 and non-glycosylated Mr = 22,000 polypeptide.     

Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons.

Glyoxysomes were isolated from etiolated cotyledons of cucumber seedlings. After separation of matrix proteins from the glyoxysomal membranes, enzymes were solubilized from the membranes by 100 mm MgCl₂ and purified by sedimentation velocity centrifugation, ion exchange chromatography, and separation on hydroxylapatite. Malate synthase, citrate synthase, and malate dehydrogenase the three enzymes of the glyoxylate cycle which were primarily membrane bound in this type of microbody-were thus obtained in a homogeneous form, as judged by sodium dodecyl sulfate-gel electrophoresis. Enzymatically active malate synthase, as obtained by solubilization of membrane proteins, behaved on Sepharose 6B columns as a protein with a molecular weight of about 70,000 and is characterized by an acidic isoelectric point. Malate synthase aggregates in the presence of Mg²⁺ and glyoxylate, yielding an active octamer with an alkaline isoelectric point and a molecular weight of about 540,000. Upon sodium dodecyl sulfate-gel electrophoresis, a subunit molecular weight of 63,000 was estimated. Citrate synthase exists as a dimer (molecular weight of 100,000) and tetramer (molecular weight of 200,000) and exhibits the same subunit molecular weight as the liver enzyme (46,000). Malate dehydrogenase was found to have a molecular weight similar to the microbody catalase (about 225,000), while for the single peptide chain a value of approximately 34,000 was determined.     

Detergent solubilized and molecular weight estimation of tumor specific surface antigen from SV40 virus transformed cells.

Mild detergent treatment of SV40 transformed mouse embryo fibroblasts, followed by (NH₄)₂SO₄ precipitation of solubilized proteins and rate zonal centrifugation in a sucrose gradient, provided direct and rapid determination of the sedimentation coefficient (4S) of tumor specific surface antigen(s) (TSSA), as detected by an antibody dependent, cell lysis assay. A molecular weight range of 50,000–60,000 was estimated for the TSSA. These results agree with those also obtained from Sephadex G-150 exclusion chromatography.     

Hydrodynamic characterization of the size and shape of atropinesterase from Pseudomonas putida

Atropinesterase from Pseudomonas putida has been investigated by means of different ultracentrifugation methods under native and denaturing conditions. The following quantities were determined: sedimentation coefficient, translational diffusion and friction coefficient, partial specific volume and molecular weight. From these data the size, shape and hydration of the enzyme molecule in solution were estimated. The results suggest that atropinesterase is a globular protein which consists of a single polypeptide chain with a molecular weight of about 30,000. In solution under non-denaturing conditions, it occurs mainly as a dimer which hydrodynamically behaves as a rigid impenetrable particle. Calculations based on the spheroid model indicate that this particle resembles a hydrated sphere with a diameter of 6.1 +/- 0.2 nm and a hydration of 0.4 +/- 0.1 g of H2O/g of protein rather than a significantly less hydrated ellipsoid of revolution. Under denaturing conditions dissociation into monomers takes place. The effects of sodium dodecyl sulphate (SDS) on size and shape suggest that dimerization results from side-by-side association of two elongated monomers rather than from end-to-end association. Approximately 57 molecules of SDS are bound per dimer before dissociation occurs concomitant with the additional binding of about 19 molecules of detergent.