Site Of ATP Hydrolysis Research Articles

Effect of streptozotocin-induced diabetes on phosphodiesterase activity in rat luteal cells.

The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca2+ and calmodulin. Nevertheless, the Ca(2+)-calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K(m) values were found for PDE from groups C, D, and I, whereas the Vmax2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats.

Binding and Hydrolysis of TNP-ATP by Escherichia coli F1-ATPase

It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated.

Conformational changes of the ATP-binding pocket of myosin and muscle contraction.

Myosin is one of the key protein components of biological force generating systems. The globular head region of the myosin molecule, subfragment-1 (S-1), contains the site for ATP binding and hydrolysis and the site for actin binding. Although the open and closed conformational states of the ligand-binding pocket have been well established with various proteins other than myosin, such an operation of the ATP-binding pocket of S-1 is purely speculative at present. A hydrophobic fluorescent dye was found to bind to the ATP-binding pocket of S-1. Fluorescence study reveals that the ATP-binding pocket of S-1 is closed and/or tightened during ATP hydrolysis.

The DNA binding domain of the vaccinia virus early transcription factor small subunit is an extended helicase-like motif.

The vaccinia virus early transcription factor (VETF) is an ATP-dependent activator of the early class of viral genes. VETF is a heterodimeric protein that binds an initiator-like element surrounding the start site of transcription. Previous studies indicated that the small subunit of VETF contacts the promoter DNA. We have taken a mutational approach to determine sequences in the VETF small subunit that are important for DNA binding. Two types of sequences were targeted for mutation: ones resembling motifs that are conserved in the nucleic acid helicase family and positively charged residues in predicted alpha-helices. Mutations affecting transcription activation were clustered in two regions. One mutation that impaired DNA binding is located near the N-terminus within the putative ATP-binding pocket that comprises helicase domain I. DNA binding was also severely reduced by mutations in a sequence resembling helicase domain VI and two putative alpha-helices that flank this domain in the C-terminal third of the polypeptide. These results indicate that the DNA binding domain in the small subunit of VETF is not isolated within a separable domain as is the case with most transcription factors, but rather, spans most of the length of the 637 residue polypeptide. A model for VETF structure is suggested in which the active site for ATP hydrolysis is integrated within an extended DNA-binding domain such that the structure and function of each domain influences that of the other.

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The site of interaction of dibucaine with the Ca(2+)-ATPase of rabbit sarcoplasmic reticulum, an ion-transporting membrane protein, was investigated by determining the effect of dibucaine on the denaturation of the transmembrane domain and the aqueous domain containing, respectively, the high-affinity Ca2+ binding sites and the site of ATP hydrolysis. In the absence of Ca2+, a single irreversible denaturation transition with Tm approximately equal to 49 degrees C is observed for the Ca(2+)-ATPase by differential scanning calorimetry (DSC). In the presence of Ca2+, but not Mg2+, Sr2+, or Ba2+, a new high-temperature transition is observed that has been shown to be due to stabilization of the transmembrane region [Lepock, J. R., Rodahl, A. M., Zhang, C., Heynen, M. L., Waters, B., & Cheng, K. H. (1990) Biochemistry 29, 681-689]. The maximum stabilization corresponds to a shift in Tm of 13.8 degrees C, and Hill analysis indicates that the Ca2+ binding site yielding stabilization has a Kd = 2.5 x 10(-4) M with a cooperativity (n) of 1. Thus, stabilization is due to Ca2+ binding not to the high-affinity sites but to one of the previously observed sites of low or intermediate affinity, which must be located in the transmembrane or stalk subdomains. Dibucaine has little effect on the Tm of the aqueous domain, but it decreases the Tm of the transmembrane domain with Kd approximately equal to 4.1 x 10(-4) M and a cooperativity of approximately 1.6, implying that destabilization is due to the binding of dibucaine to sites of intermediate or moderately high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic cooperativity in the Ca 2+-dependent ATPase activity of spinach chloroplast coupling factor (CF 1)

The Ca 2+-ATPase activity of CF 1, the soluble chloroplast coupling factor from spinach chloroplasts, was measured over ATP concentrations between 0.05 μM and 1000 μM. At very low ATP concentrations (<1 μM), CF 1 exhibited a high affinity, low capacity mode of catalysis ( K m = 0.05 μM, V max = 1 nmol min −1 mg −1). This activity was unaffected by the ϵ-subunit of CF 1, and was stimulated 2–3-fold by tentoxin or by anti-CF 1 antibody. All three of these agents inhibit Ca 2+-ATPase at higher ATP concentrations. The pattern of ATP and ITP stimulation of [ 32P]P i release from [γ- 32P]ATP, over the entire range of ATP concentrations studied, was investigated by mathematical modelling. Simulation of the observed data was possible only if three catalytic sites were operative, ruling out models for CF 1 turnover with only two catalytic sites for ATP hydrolysis (whether or not a solely regulatory site was also present). Cooperativity between catalytic sites was also required for simulation of the data. The resultant mathematical model for ATP hydrolysis can most simply be accommodated using a structural model for CF 1 in which three equivalent sites operate alternately during turnover (Boyer, P.D. (1989) FASEB J. 3, 2164–2178). We cannot, however, rule out a model in which an asymmetrical enzyme is maintained throughout turnover, providing that the catalytic sites in such an enzyme interact cooperatively during turnover.

Reconstitution of recombinant 40-kDa subunit of the clathrin-coated vesicle H(+)-ATPase.

We have proposed a model of the ATP hydrolytic sector of the clathrin-coated vesicle H(+)-ATPase wherein significant catalysis requires four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have cloned and expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity. This subunit lacks ATP hydrolytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subunit is required for vacuolar-type proton pump function.

The vacuolar ATPase of Neurospora crassa.

The filamentous fungus Neurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8-10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V1 and an integral membrane complex called V0. Genes encoding three of the subunits have been isolated. vma-1 and vma-2 encode polypeptides homologous to the alpha and beta subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis. vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.

ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (Aα, Aβ, and Aγ) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained Aβ and Aγ HCs, while the Aα HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887–895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897–901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 μg/ml thermolysin for 45 min. we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5α, 5β, 6α, and 6β). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained Aβ or Aγ HC. Thermolysin digests, TH 1, 2, 5α, and 6β were found to be derived from Aβ HC, while TH 3, 4, 5β and 6α originated in the Aγ HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the Aβ and Aγ HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.

Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A. L., and Nikaido, H. (1990) J. Biol. Chem. 265, 4254-4260). A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside. Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside. The purified proteins catalyze both maltose transport activity and ATP hydrolysis. In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000. Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis. Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK.

Influence of ATP turnover and metabolite changes on IMP formation and glycolysis in rat skeletal muscle

Deamination of AMP to inosine monophosphate (IMP) and NH3 is thought to be regulated by the observed increases in ADP, AMP, and H+. We have examined this hypothesis by comparing the rate of IMP accumulation in contracting and noncontracting rat skeletal muscle. The rate of IMP formation was high during ischemic contraction, and consistent with previous studies, formation of IMP was associated with high levels of muscle lactate, depletion of phosphocreatine (PCr), and increased levels of ADP and AMP. When the contraction period was followed by 5-min anoxic recovery, the metabolic changes were maintained, but no further IMP or lactate was formed. During long-term (2-4 h) anoxia, the rate of IMP formation was less than 4% of that during contraction, despite similar changes in PCr, lactate, ADP, and AMP. It is concluded that the observed changes in the intracellular chemical environment are not sufficient to explain the high rate of IMP formation during contraction but that a combination of metabolic stress and a high ATP turnover rate is required. It is suggested that a high ATP turnover rate during conditions of metabolic stress results in transient increases in ADP and AMP at the site of ATP hydrolysis and that these activate AMP deaminase and glycolysis. An alternative hypothesis is that these processes are regulated by the increase in cytosolic Ca2+ in a contracting muscle.

Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae.

Subunit a of the vacuolar membrane H(+)-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae contains a catalytic site for ATP hydrolysis. N-terminal sequences of six tryptic peptides of the subunit were determined. Based on the peptide sequence information, a 39-base oligonucleotide probe was synthesized, and the gene encoding the subunit (VMA1) was isolated from a genomic DNA library by hybridization. The nucleotide sequence of the gene predicts a polypeptide of 1,071 amino acids with a calculated molecular mass of 118,635 daltons, which is much larger than the value 67 kDa estimated on sodium dodecyl sulfate-polyacrylamide gels. N- and C-terminal regions of the deduced sequence (residues 1-284 and 739-1,071) are very similar to those of the catalytic subunits of carrot (69 kDa) and Neurospora crassa (67 kDa) vacuolar membrane H(+)-ATPases (62 and 73% identity over 600 residues, respectively). The homologous regions also show about 25% sequence identity over 400 residues with beta-subunits of F0F1-ATPases. In contrast, the internal region containing 454 amino acid residues (residues 285-738) shows no detectable sequence similarities to any known ATPase subunits and instead is similar to a yeast endonuclease encoded by the HO gene. None of the six tryptic peptides is located in this internal region. Northern blotting analysis detected a single mRNA of 3.5 kilobases, indicating that the gene has no introns. Although the reason for the discrepancy in molecular mass is unclear at present, these results suggest that a novel processing mechanism, which might involve a post-translational excision of the internal region followed by peptide ligation, operates on the yeast VMA1 product. The VMA1 gene has proven to be the same gene as the TFP1 gene (Shih, C.-K., Wagner, R., Feinstein, S., Kanik-Ennulat, C., and Neff, N. (1988) Mol. Cell. Biol. 8, 3094-3103) whose dominant mutant allele (TFP1-408) confers a dominant trifluoperazine resistance and Ca2(+)-sensitive growth. This and our findings suggest that the vacuolar membrane H(+)-ATPase participates in maintenance of cytoplasmic Ca2+ homeostasis.

Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme.

The molecular composition and organization of the row of axonemal inner dynein arms were investigated by biochemical and electron microscopic analyses of Chlamydomonas wild-type and mutant axonemes. Three inner arm structures could be distinguished on the basis of their molecular composition and position in the axoneme as determined by analysis of pf30 and pf23 mutants. The three inner arm structures repeat every 96 nm and are referred to here as inner arms I1, I2, and I3. I1 is proximal to the radial spoke S1, whereas I2 and I3 are distal to spokes S1 and S2, respectively. The mutant pf30 lacks I1 whereas the mutant pf23 lacks both I1 and I2 but has a normal inner arm I3. Each of the six heavy chains that was identified as an inner dynein arm subunit has a site for ATP binding and hydrolysis. Two of the heavy chains together with a polypeptide of 140,000 molecular weight form the inner arm I1 and were extracted from the axoneme as a complex that had a sedimentation coefficient close to 21S at high ionic strength. Different subsets of two of the remaining four heavy chains form the inner arms I2 and I3. These arms at high ionic strength are dissociated as 11S particles that include one heavy chain, one intermediate chain, two light chains, and actin. These and other lines of evidence indicate that the inner arm I1 is different in structure and function from the inner arms I2 and I3.

Inhibition of Mitochondrial F1-ATPase Activity by an Anti-α Subunit Monoclonal Antibody Which Modifies Interactions Between Catalytic and Regulatory Sites

A monoclonal antibody, 7B3, specific to the alpha subunit of the mitochondrial ATPase-ATP synthase inhibited the rate of ATP hydrolysis by either soluble F1 or electron transport particles up to a maximum of 75%. However, 7B3 did not modify the rate of ITP hydrolysis. In addition, the apparent Km for MgATP extrapolated at high ATP concentrations had the same value in the absence as in the presence of 7B3. The antibody did not change the inactivation rate of F1-ATPase induced by dicyclohexylcarbodiimide or 4-chloro-7-nitro-2,1,3-benzoxadiazole. These observations indicate that 7B3 did not directly interfere with the catalytic sites of ATP or ITP hydrolysis. On the contrary, 7B3 modified the interaction between nucleotide sites and therefore the regulation of the rate of ATP hydrolysis. Indeed, 7B3 changed into a positive cooperativity the negative cooperativity observed when measuring the rate of ATP hydrolysis as a function of ATP concentration. 7B3 also increased the binding of ADP to F1. 7B3 prevented the rapid phase of inactivation of F1 by 5'-p-fluorosulfonylbenzoyladenosine. This phase has been correlated to the binding of 5'-p-fluorosulfonylbenzoyladenosine to regulatory sites (Di Pietro, A., Godinot, C., Martin, J. C., and Gautheron, D. C. (1979) Biochemistry 18, 1738-1745). The inhibition of ATP hydrolysis is concomitant with the binding of 1 mol of IgG or of 2 mol of Fab fragments per mol of F1. However, by further increasing the ratio Fab/F1, only 1 mol of Fab remained bound to F1 without change in inhibition of ATPase activity. All these experiments strongly support the suggestion that F1 conformational changes occurring upon binding of 7B3 to alpha subunit induce a modification of interactions between nucleotide sites. This modification would be consecutive to a change in the normal interaction between the alpha and beta subunits which is required to observe an active rate of ATP hydrolysis or synthesis. In conclusion, the use of this monoclonal antibody demonstrates for the first time in mammalian F1 the role of the conformation of the alpha subunit in the regulation of the ATPase activity.

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1.

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases.

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.

Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains.

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.

Electron microscopic study on the location of 23 kDa and 50 kDa fragments in skeletal myosin head.

The functional activities of myosin head are located in a 95 kilodalton (kDa) heavy chain which can be divided into three fragments of 23 kDa, 50 kDa, and 20 kDa. ATP hydrolysis sites were suggested to be located in the 23 kDa and 50 kDa fragments, and actin binding sites were in the 50 kDa and 20 kDa fragments. In this study, we obtained electron microscopic images of the myosin molecule bound with antibodies directed to the 23 kDa and 50 kDa fragments. We determined that the antigenic sites for 23 kDa fragment are located at 140-180 A from the head-rod junction of myosin, and those for 50 kDa fragment at 160 A from the junction and at the tip of the head itself. The relationship between the spatial locations and the primary structures is discussed.

Inhibition of the H +-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol Evidence for a tight coupling between ATP hydrolysis and proton translocation

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100μM) and proton pump activity ( I 50⋍15 μM, complete at 100 μM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg 2+-ATPase activity, but not proton pumping, was partially restored by N, N′-dicyclohexylcarbodiimide,indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H +-ATPase.