Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

Restoration of coupling factor activity to Escherichia coli ATPase missing the delta subunit.

We separated the two minor subunits (δ and ε) of the $\text{E. coli}$ ATPase from the major subunits (α, β, and γ). The minor subunit fraction was obtained by treating purified ATPase with pyridine following the procedure that Nelson $\text{et al}$. (J. Biol. Chem. 348, 2049 [1973]) used to separate the subunits of chloroplast ATPase. The minor subunit fraction restored the capacity of ATPase lacking the delta subunit to recombine with ATPase-depleted membrane vesicles and to reconstitute energy coupling to the transhydrogenase and oxidative phosphorylation in the vesicles. These results clearly implicate the delta subunit in the attachment of the ATPase to the membrane.     

Release and purification of micrococcus lysodeikticus ATPase from membrane extracted with n-butanol

The ATPase associated with the membranes of Micrococcus ysodeikticus has been released into the aqueous phase (i.e. solubilized) by extracting the membranes with $\text{n-}\text{butanol}$ in a two-phase system modified from the procedure of Maddy, A.H. (164) Biochim. Biophys. Acta 88, 448–449. A procedure for the release and purification of the ATPase from the membranes extracted with $\text{n-}\text{butanol}$ is described as an alternate method to that previously used for the shock-wash ATPase. Upon extracting the membrane suspensions with $\text{n-}\text{butanol}$ the soluble ATPase released into the buffer phase no longer exhibits stimulation by trypsin in contrast to the shock-wash type of ATPase. As shown by Salton, M. R. J. and Schor, M. T. (1972) Biochem. Biophys. Res. Commun. 49, 350–357, the shock-wash ATPase possesses associated protein(s) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis whereas these are absent from the purified ATPase released by the $\text{n-}\text{butanol}$ method. The specific activities of the purified ATPase released by the two methods were generally similar, the $\text{n-}\text{butanol}$ type being consistently somewhat higher.     

Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin.

Photoaffinity labelling of tryptic and chymotryptic heavy meromyosin with 3′O-3-[N-(4-azido-2-nitrophenyl) amino]propionyl-adenosine 5′-triphosphate (arylazido-β-alanine ATP) resulted in incorporation of radioactivity and inhibition of the ATPase activity. ATP prevented the reaction with the photoaffinity label, as shown by the lack of incorporation of ³H and intact ATPase activity. On the tryptic digestion of either type of photoaffinity labeled HMM the label was found in a 25K peptide identifiable with the N-terminus of the myosin heavy chain (Lu et al., Fed. Proc. $\text{37}$ 1695 1978). The results are discussed in the light of previous localization of the reactive thiol groups, SH-1 and SH-2 (Balint et al., Arch. Biochem. Biophys. $\text{190}$, 793 1978).     

Role of Mg2+ ions in the subunit structure and membrane binding properties of bacterial energy transducing ATPase.

ATPase extracted from $\text{Streptococcus faecalis}$ membranes was purified by preparative slab gel electrophoresis in the presence of Mg⁺⁺ (plus Mg²⁺ ATPase) and without Mg²⁺ (minus Mg²⁺ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg²⁺ ATPase had 5 types of subunits (αβγδ?) and reattached normally to depleted membranes. The minus Mg²⁺ ATPase had the αβγ and ? chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg²⁺ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg²⁺ ATPase the data are consistent with the subunit stoichiometry and arrangement, (α₃β₃ γ ?)-Mg²⁺)_n?(δ).     

Specificity of association of a Ca2+/Mg2+ ATPase with cholinergic synaptic vesicles from Torpedo electric organ.

Cholinergic synaptic vesicles from the electric organ of $\text{Torpedo}$$\text{californica}$ have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase in the cholinergic synaptic vesicle.     

Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin

The human erythrocyte membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase responded to the presence of an acidic phospholipase A₂ and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the $\text{Ca}{}^{\mathrm{2+}}\text{M}{}^{\mathrm{2+}}$ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.     

The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli

Removal of the F₁ ATPase from membrane vesicles of $\text{Escherichia}\text{coli}$ resulted in leakage of protons across the membrane through the F_O portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F₁ ATPase to F₁-stripped everted membrane vesicles. It is concluded that in F₁-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F₁ ATPase.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Pyrenesulfonyl azide: a covalent probe permitting in vitro desensitization of labeled acetylcholine-rich membrane fragments from Torpedo californica.

Acetylcholine receptor-rich membrane fragments from $\text{Torpedo}\text{californica}$ electroplax after covalent labelling at the protein-lipid boundary by nitrenes generated $\text{in}\text{situ}$ from pyrenesulfonyl azide can bind $\text{[}{}^{125}\text{I]-α-bungarotoxin}$. The covalent attachment of 6–8 molecules of the fluorescent probe/receptor molecule also does not perturb the marked effect on the rate of α-bungarotoxin binding to electroplax membranes exerted by their preincubation with carbamylcholine. This phenomenon, which is analogous to pharmacological desensitization of receptors in synaptic junctions, is fully reversible upon removal of carbamylcholine (Quast, V., Schmerlik, M., Lee, T., Witzemann, V., Blanchard, V. and Raftery, M.A. (1978) Biochemistry $\text{17}$, 2405–2414). Torpedo electroplax membranes, whether tagged with the covalent probe or freshly isolated, regain the original fast rate of α-bungarotoxin binding upon dilution of carbamylcholine.     

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Three new alpha-chains of collagen from a non-basement membrane source

Three new collagen α chains have been isolated from synovial membrane, gingiva and skin. Two of these have a similar chromatographic behaviour to the α[A] and α[B] chains described by Burgeson $\text{et al}$. (4) from a foetal basement membrane source but have been separated from another contaminating α chain, α[C]. The α[A] and α[B] chains are present in approximately equal amounts. They contain no detectable 3-hydroxyproline, are highly glycosylated and all sugar residues are present as the disaccharides. The percentage of hydroxylation of the lysine is of the order of 70%. Only a third of these hydroxylysine residues are glycosylated. The significance of these peptides, present in a tissue substantially free of basement membrane, is discussed.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. $\text{N,N′-}\text{Dicyclohexylcarbodiimide}$, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, $\text{N-1-}\text{napthylphthalamate}$, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H⁺ transport across their respective membranes.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.