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The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

The tubulin dimer contains two guanine nucleotide binding sites, a nonexchangeable site occupied by GTP and an exchangeable site (E-site) occupied by GTP or GDP. Under the conditions used in this study the E-site GTP was hydrolyzed at a rate of 8 x 10(-5) s(-1) at 37 degrees C. This rate is stimulated four- to fivefold by nocodazole, an antimitotic drug. We studied the characteristics of this drug-stimulated reaction to learn more about the hydrolytic center of tubulin. The reaction, studied using single turnover kinetics, i.e., in the absence of added GTP, has a pH optimum of 6.6 to 7.2 and an activation energy of 88 kJ x mol(-1). It is first-order with respect to tubulin-GTP, indicating that hydrolysis is not dependent on an aggregation process. Divalent cations stimulate the hydrolysis three- to six-fold over the rate in the presence of EDTA. The reaction has a requirement for Na+ that is not satisfied effectively by other monovalent cations. In contrast, Na+ and K+ are almost equally effective in the tubulin assembly reaction. Different purine nucleoside triphosphates can bind to the E-site and are hydrolyzed. GTP and ITP are hydrolyzed at equivalent rates and XTP and ATP are hydrolyzed at a rate about half as fast. Hydrolysis is inhibited by the reagents diethylpyrocarbonate and N-ethylmaleimide, although the GTPase activity is less sensitive than the assembly reaction. The reaction rate in D2O is twice the rate in H2O. This inverse isotope effect suggests the involvement of a sulfhydryl group in the rate-limiting step in the reaction.

After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.