The aminoacyl-tRNA synthetase-tRNA complex: detection by differential labelling of lysine residues involved in complex formation

The interaction of tRNA^Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by differential acetylation of lysine residues. The synthetase was trace-labelled in the free form and as the synthetase-tRNA^Tyr complex with [³H]acetic anhydride. In a second step the two ³H-labelled enzyme preparations were fully acetylated with cold reagent under denaturing conditions and were mixed with synthetase that had been homogeneously labelled with excess [¹⁴C]acetic anhydride. Peptides containing labelled lysine residues were isolated after chymotryptic digestion and their ${}^{14}\text{C}{}^3\text{H}$ ratios were determined. These ratios reflect the reactivity of primary amino groups towards acetic anhydride.Involvement of lysine side-chains in complex formation with tRNA^Tyr was suggested from altered ${}^{14}\text{C}{}^3\text{H}$ ratios. Out of the 22 primary amino groups of tyrosyl-tRNA synthetase at least three showed reduced reactivities towards acetic anhydride in the synthetase-tRNA^Tyr complex by factors of 1.6, 1.9 and 6.8, respectively. The sequences around these lysine residues have been determined enabling their placement when the primary and tertiary structure of the enzyme are available (G. L. E. Koch, to be published). No lysine residue of increased reactivity in the synthetase-tRNA^Tyr complex has been detected.Only one molecule of tRNA^Tyr binds to the dimeric synthetase molecule under the conditions of the differential labelling. If the binding site for the tRNA is on one of the two identical subunits, any observed decrease in chemical reactivity of a particular lysine residue should not exceed a factor of two. The detection of a lysine residue which reacts about seven times more slowly in the synthetase-tRNA complex could therefore indicate that the single binding site is formed by both enzyme subunits.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Thymidine accumulation by choroid plexus in vitro

In vitro, the accumulation and release of [methyl-³H]thymidine ([${}^3\text{H}$]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [${}^3\text{H}$]thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the [${}^3\text{H}$]thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm [${}^3\text{H}$]thymidine in the medium, the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient. However, the majority of the [${}^3\text{H}$]thymidine within the choroid plexus was metabolized to [${}^3\text{H}$]thymidine nucleotides at low extracellular [${}^3\text{H}$]thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration.     

Studies on (K+ + H+)-ATPase: VI. Determination of the molecular size by radiation inactivation analysis

(1) A $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ to basal Mg²⁺-ATPase activity from 9 to 20. (2) The target size of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4·10¹¹ is valid for membrane-bound ATPases. (3) The target size of $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K⁺-stimulated $\text{p-}\text{nitrophenylphosphatase}$ activity has a target size of 295 kDa. (4) In the presence of added Mg²⁺ the target sizes of the $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and its phosphatase activity are decreased by about 15%, while that for the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is not significantly changed. This observation is discussed in terms of a Mg²⁺-induced tightening of the subunits composing the $\text{(}\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule.     

Thin-layer mapping of peptides labeled with 3H or 14C via reductive methylation.

Two dimensional tryptic peptide maps have been obtained from 2 μg (40 pmol) of protein digest following labeling with ³H or ¹⁴C via reductive methylation. A simple labeling procedure is complete within 1 h; autoradiographs of ¹⁴C-labeled maps and fluorographs of ³H-labeled maps are obtained in 72 and 24 h, respectively. Tryptic peptide maps of ${}^{14}\text{C}{}^3\text{H}$ methylated α- and β-tubulin, and rabbit muscle, chick muscle, and chick brain actins show approximately the expected number of peptides. Methylation does not appear to measurably alter the map positions of the peptides relative to unmethylated peptides in the solvent systems used for either electrophoresis or chromatography.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

31P and 17O NMR of Mn(III)-containing acid phosphatase: Evidence for direct metal-phosphate interaction and oxygen exchange from water into inorganic phosphate

The acid phosphatase isolated from sweet potato tubers by us is unique Mn(III)-containing enzyme which hydrolyzes phosphomonoesters and nucleotide phosphates. The present ³¹P and ¹⁷O NMR studies of the Mn(III)-containing acid phosphatase solved two important problems. The broadening of the phosphate ³¹P resonance signal in the 1:1 enzyme-substrate system shows evidence for direct metal-phosphate interaction in the Mn(III)-containing acid phosphatase. In addition, the ¹⁷O NMR evidence for oxygen exchange from water into inorganic phosphate strongly indicates that the Mn(III)-containing acid phosphatase catalyzes an apparent transition state displacement and P-O cleavage as follows: $\text{ROPO}{}_3{}^{\mathrm{=}}\text{+ H}{}^{17}\text{OHROH + H}{}^{17}\text{OPO}{}_3{}^{\mathrm{=}}$.     

Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide

The $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rabbit sarcoplasmic reticulum, when labelled at two Ca²⁺-protected sites with $\text{N-}\text{cyclohexyl}\text{-N′-(4-}\text{dimethylamino}\text{-α-}\text{naphthyl}\text{)}\text{carbodiimide}$ (NCD-4) retains Ca²⁺ binding capacity at the sites with $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values of approx. 3 μM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca²⁺ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca²⁺. It retains the ability to form phosphoenzyme, and can most likely translocate Ca²⁺.     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

The interaction of |C_nH_2n+1N⁺(CH₃)₃| · I^? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by ³¹P-NMR spectroscopy. It is shown that the effective anisotropy of ³¹P chemical shift (?Δσ_eff) of the lamellar phospholipid liquid-crystalline phase L_α increases with increasing concentration and alkyl chain length of the drug. Addition of $\text{|}\text{C}{}_6\text{H}{}_{13}\text{N}{}^{\mathrm{+}}\text{(CH}{}_3\text{)}{}_3\text{| ·I}{}^{\mathrm{?}}$ or |C₉H₁₉N⁺(CH₃)₃|·I^? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.     

Relationship between H+, anion, and monovalent cation movements and Ca2+ transport in sarcoplasmic reticulum: further proof of a cation exchange mechanism for the Ca2+-Mg2+-ATPase pump

Two spectroscopic probes of free internal Ca²⁺ were used to determine the influence of H⁺ and anion permeation on the active transport of Ca²⁺ by skeletal sarcoplasmic reticulum. The studies were carried out on a well-characterized Ca²⁺-Mg²⁺-ATPase-rich sarcoplasmic reticulum fraction. Studies of D. McKinley and G. Meissner (1977, FEBS Lett., 82, 47–50) show that this fraction consists of two populations of vesicles: type I which has an electrically active monovalent cation (M⁺) permeability and type II which lacks it. The present study distinguishes between electrically active (charge-carrying) and electrically silent (e.g., countertransport) mechanisms of ion permeation in the two vesicles and shows how the active transport of Ca²⁺ is influenced by these permeabilities. The major results are as follows: (1) Both type I and II vesicles have an electrically active H⁺ permeability. (2) Type I vesicles have electrically active anion (A^?) permeabilities; type II vesicles do not. (3) At low concentrations of nonpenetrating buffers, ion imbalances across the membrane can create pH imbalances. This is due to the coupling of M⁺ and A^? movements with H⁺ movements. Following a jump in KCl concentration internal acidification is observed in type I vesicles while internal alkalinization is observed in type II vesicles. These pH gradients are dissipated on a time scale of seconds and tens of minutes for type I and II vesicles, respectively. (4) Tris(hydroxymethyl)aminomethane (Tris) was shown to be effective in dissipating pH gradients in type II vesicles. A model is proposed whereby HCl is equilibrated across the membrane by a Tris-catalyzed transport cycle involving transport of an ion pair between Tris-H⁺ and Cl^? and return of the unprotonated form of the buffer. (5) The permeabilities of several physiological and nonphysiological anions were determined for type I and II vesicles. Electrically active permeability was demonstrated for Cl^? and phosphate in type I vesicles. Type II vesicles lacked electrically active mechanisms for these two anions. Evidence is given for slow $\text{Cl}{}^{\mathrm{?}}\text{OH}{}^{\mathrm{?}}$ exchange and for rapid $\text{Cl}{}^{\mathrm{?}}\text{HCO}{}_3{}^{\mathrm{?}}$ exchange in type II vesicles. Electrically silent phosphate influx probably occurs by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}\text{OH}{}^{\mathrm{?}}$ exchange. (6) Under normal conditions the Ca²⁺ uptake of type II vesicles is masked. It can be unmasked by addition of nigericin in the presence of Tris. The combination of ionophore and penetrating buffer render the type II vesicles KCl permeable, allowing the replenishment of internal K⁺ during active transport. The results are analyzed and shown to be in agreement with the Ca²⁺-Mg²⁺-ATPase pump acting as a $\text{Ca}{}^{\mathrm{2+}}\text{K}{}^{\mathrm{+}}$ exchanger. The results are shown to be in disagreement with electrogenic models of pump function.     

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of $\text{ATP}\text{10}{}^6$ cells were observed; a maximum of 3.2 nmoles of $\text{ATP}\text{10}{}^6$ cells was reached at 12 h (G 2 phase). The adenylate energy charge, $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.     

The kinetics of myofibrillar protein breakdown in perfused rat skeletal muscle

N^t-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with $\text{[}{}^{\mathrm{Me}}\text{-}{}^3\text{H}\text{]}\text{methionine}$. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ with uptake of 1 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K⁺ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c₂-oxidoreductase showed a pumping activity of 0.9 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Ca2+-cardiolipin interaction in a model system Selectivity and apparent high affinity

The interaction of cardiolipin with Ca²⁺ was assessed by measuring the cardiolipin-mediated extraction of ⁴⁵Ca²⁺ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca²⁺ with high affinity [$\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(}\text{apparent}\text{)=0.70±0.17 μ}\text{M (S.D.)}$]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca²⁺ is insensitive to Na⁺, but is inhibited by divalent cations with $\text{Mn}{}^{\mathrm{2+}}\text{>}\text{Zn}{}^{\mathrm{2+}}\text{>}\text{Mg}{}^{\mathrm{2+}}$. In addition La³⁺ and Ruthenium red are particularly potent inhibitors of Ca²⁺ binding by cardiolipin. Cardiolipin-mediated extraction of Ca²⁺ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca²⁺-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.