Kinetic studies of ATP synthase: The case for the positional change mechanism

The mitochondrial ATP synthases shares many structural and kinetic properties with bacterial and chloroplast ATP synthases. These enzymes transduce the energy contained in the membrane's electrochemical proton gradients into the energy required for synthesis of high-energy phosphate bonds. The unusual three-fold symmetry of the hydrophilic domain, F₁, of all these synthases is striking. Each F₁ has three identical subunits and three identical subunits as well as three additional subunits present as single copies. The catalytic site for synthesis is undoubtedly contained in the subunit or an , interface, and thus each enzyme appears to contain three identical catalytic sites. This review summarizes recent isotopic and kinetic evidence in favour of the concept, originally proposed by Boyer and coworkers, that energy from the proton gradient is exerted not directly for the reaction at the catalytic site, but rather to release product from a single catalytic site. A modification of this binding change hypotheses is favored by recent data which suggest that the binding change is due to a positional change in all three subunits relative to the remaining subunits of F₁ and F₀ and that the vector of rotation is influenced by energy. The positional change, or rotation, appears to be the slow step in the process of catalysis and it is accelerated in all F₁F₀ ATPases studied by substrate binding and by the proton gradient. However, in the mammalian mitochondrial enzyme, other types of allosteric rate regulation not yet fully elucidated seem important as well.     

Studies on lens proteins of mice with hereditary cataract. I. Comparative studies on the chemical and immunochemical properties of the soluble proteins of cataractous and normal mouse lenses

Total soluble and insoluble proteins of the lens were similar in normal and hereditary cataractous mice up to 1 week of age. Thereafter, the normal mouse lens showed a continued increase in weight and protein content until 500 days of age. In cataractous mice, while the total protein content increased up to 60 days and reached a plateau, the soluble protein content declined dramatically from day 22 to day 60, and then the rate of decrease remained constant up to 500 days.At different ages, the soluble proteins were separated by gel filtration into the high molecular weight proteins, α-, β- and γ-crystallin fractions. All of these showed an age-related increase in the normal lens, and the relative values of α- and β-crystallins increased for a 410-day period. On the other hand, in the cataractous process, the high molecular weight protein increased, and α-, β- and γ-crystallins decreased: the degree was especially marked in γ-crystallin.Immunochemical studies indicated that the aggregation of β-crystaUin occurred much earlier in the cataractous lens than in the normal. Analysis of the amino acid composition and ultraviolet absorption spectre revealed no significant chemical differences between the crystallins of the normal and the cataractous lens.     

An immunochemical aid to sequence determination of proteins.

A specific radioimmunoassay for peptides has been developed using ¹²⁵I-labeled peptides and a double-antibody precipitation. Cross-reacting peptides are measured by inhibition of the binding of the labeled cyanogen bromide peptide to its antibody. The assay, which allows detection of picomole quantities, was used to monitor the purification of two overlapping tryptic peptides from a complex mixture of peptides. These were shown to contain a portion of the sequence of the radio-labeled cyanogen bromide peptide and a portion of the sequence of a cyanogen bromide peptide which follows in the polypeptide chain. The need to analyze many fractions in a digest in order to locate a desired peptide is thus avoided. The general suitability of this method for the purification of specific peptides from digestion mixtures of other large proteins is discussed.     

Purification and immunochemical characterization of malate synthase from Euglena gracilis.

A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).     

Hemangiopericytoma in a male breast. Report of a case with cytologic, histologic and immunochemical studies

A hemangiopericytoma in a male breast was studied by fine needle aspiration (FNA) biopsy. The FNA smears contained tissue clumps showing knob-like formations of atypical cells, spindle-shaped cells and fragments of capillaries lined by normal endothelial cells. Immunocytochemical study showed a positive reaction for vimentin, but a negative reaction for desmin and keratin. Staining for Factor VIII was positive only in the capillaries and endothelial cells. The cytodiagnosis was "mesenchymal tumor." Histopathologic study of the mastectomy specimen made the final diagnosis of hemangiopericytoma. While FNA cytology and immunocytochemistry cannot make a definitive diagnosis of this rare vascular tumor, they can be decisive in planning the surgical treatment, as in the present case.     

Molecular-size standards for use in radiation-inactivation studies on proteins.

The accuracy of the radiation-inactivation technique for estimating molecular size was investigated with a range of proteins of known molecular mass. With the use of irradiation with a 16 MeV electron beam, inactivation was examined both in frozen samples at 77 K and in freeze-dried samples at room temperature. The effect of the presence of detergents and chloroplast membrane preparations was also measured. It was demonstrated that proteins added as internal standards, including malate dehydrogenase, glucose-6-phosphate dehydrogenase and cytochrome c, can provide an accurate calibration of molecular size. However, a disadvantage of the technique was that the target size of oligomeric enzymes could be that of either the monomers, dimers or higher oligomers. The detergent Triton X-100 increased the rate of inactivation of the proteins investigated.     

Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

The relationship of the active sites which catalyze the three reactions in the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthetase activity. During chemical modification with diethyl pyrocarbonate (DEPC), the three activities were inactivated at significantly different rates, indicating that at least three distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting two essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protected both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all three activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.     

Microtubular proteins of Chlamydomonas reinhardtii. An immunochemical study based on the use of an antibody specific for the beta-tubulin subunit.

An immunochemical assay for tubulin subunits is described. The method is applied directly to homogenates of Chlamydomonas reinhardtii solubilized in sodium dodecyl sulfate (Na dodecyl-SO4), and it makes use of a two-dimensional electrophoresis system; the first separation is carried out by Na dodecyl-SO4-polyacrylamide gel electrophoresis and the second by electrophoresis into an agarose gel containing antibodies. Tubulin is precipitated in the form of a "rocket" and the method is made quantitative through the use of cells labeled with [35S]sulfate. The antiserum used in this assay was prepared in rabbits using beta subunit of tubulin purified from Chlamydomonas flagella by two preparative Na dodecyl-SO4-polyacrylamide gel electrophoreses. This antiserum and an antiserum to alpha subunit of tubulin from porcine brain, prepared for comparative study, were extensively characterized. Both antisera show specificity for the polypeptide used as antigen and react with the native dimeric tubulin. The antiserum to beta subunit from Chlamydomonas flagella also forms immunoprecipitates with native brain tubulin and its beta subunit when used at high titer. In contrast, the antiserum to alpha subunit from porcine brain does not cross-react with Chlamydomonas tubulin. The immunochemical assay was applied to Chlamydomonas cells synchronized by a 12-h light/dark cycle. In cells collected during the light period (late G1), after removal of flagella, the content of tubulin is estimated to be 0.3% of total protein. As cells enter the dark period there is a striking increase in tubulin content which reaches a maximum just before cell division.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

The association of acrylamide with proteins. The interpretation of fluorescence quenching experiments

The association properties of acrylamide with a number of proteins in aqueous solution have been investigated by a fluorescence-quenching method previously used in micelles and lipid bilayers (Blatt, E., Chatelier, R.C. and Sawyer, W.H. (1984) Chem. Phys. Lett. 108, 397-400). At pH 7.0, acrylamide partitions between the bulk aqueous phase and the proteins, human serum albumin, monellin and ovalbumin. Comparison with an earlier method of analysis (Sikaris, K.A., Thulborn, K.A. and Sawyer, W.H. (1981) Chem. Phys. Lipids 29, 23-36) confirms the data quantitatively. For human serum albumin at pH 2.2, acrylamide associates according to both partition and binding processes. Equilibrium dialysis experiments performed for the latter system verify that acrylamide associates with proteins.     

Chemical and immunochemical studies on the structure of four snail galactans

The four snail galactans studied are polysaccharides of high molecular weight that are composed entirely of d- and l-galactosyl residues. AaG and CnG, which had not previously been studied, are highly branched galactans composed mainly of (1→3)- and (1→6)-linked galactosyl residues, as shown by the results of periodate oxidation and permethylation studies. On methylation, HpG, CnG, and AaG yielded ～40% of 2,3,4,6-tetra-, 40% of 2,4-di-, 7–14% of 3,4,6-tri-, and 8–12% of 2,4,6-tri-O-methylGal derivatives. BgG gave equal amounts of tetra- and di-O-methyl derivatives, and 8.5% of 2,4,6-tri-O-methylGal, and 10% was unmethylated Gal, indicating 1, 2, 3, 4, and 6 substitution not previously reported in nature. Antisera to the four galactans showed various degrees of cross-reactivity, indicating structural differences ascribable partially to determinants involving a galactose phosphate and, probably, to the linkage and the position of l-Gal in the molecule. The galactans differed in susceptibility to d-galactose oxidase, and some of the immunochemical observations are most probably attributable to species-specific differences in distribution of linear stretches and branches. The first stages of Smith degradation of HpG and AaG showed a substantial increase in unsubstituted (1→3)- and (1→6)-linked residues. These results, and the appearance of linear stretches within the native galactans preclude the strictly dichotomously-branched structure proposed earlier.     

Comparative studies on immunochemical properties of female-specific serum protein and egg yolk proteins in rainbow trout (Salmo gairdneri).

1. Female-specific serum protein (FS) and egg yolk proteins of mature female rainbow trout (Salmo gairdneri) were studied comparatively with reference to their immunological and some physicochemical properties. 2. FS was considered to be composed of two egg proteins, E1 and E2, which were isolated from the egg yolk by gel filtration. 3. The molecular weights of FS, E1 and E2 were estimated by gel filtration to be approx 600,000, 300,000 and 35,000, respectively. 4. The analysis with sodium dodecyl sulphate electrophoresis indicated that E1 consisted of two subunits with molecular weights of 90,000 and 15,000, and E2 was estimated to be a dimer of a molecule of 15,000. 5. Male and immature female trout were injected with estradiol-17 beta, and the production of proteins with the same antigenicity with FS was demonstrated.