Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13C NMR study

[$\text{N-}{}^{13}\text{CH}{}_3$] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [$\text{N-}{}^{13}\text{CH}{}_3$] phosphatidylcholine is measured with ¹³C NMR. The transbilayer movements of $\text{[N-}{}^{13}\text{CH}{}_3\text{]-}\text{dioleoyl}$ phosphatidylcholine and [$\text{N-}{}^{13}\text{CH}{}_3$] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [$\text{N-}{}^{13}\text{CH}{}_3$] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187

The antibiotic A23187 carries Ca²⁺ across Müller-Rudin membranes made from $\text{1,2-}\text{dierucoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ and $\text{n-}\text{decane}$. The conductance of the membranes is not increased by the Ca²⁺-transport. The flux depends linearly on Ca²⁺ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca²⁺-fluxes of about $\text{10}{}^{\mathrm{?10}}\text{mol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{s}{}^{\mathrm{?1}}$ were found. A counter transport of H⁺ could not be detected.The complex formation between A23187 and Ca²⁺ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Characterization of (Na+ + K+)-ATPase liposomes: I. Effect of enzyme concentration and modification on liposome size,intramembrane particle formation and Na+,K+-transport

Rabbit renal ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ (EC 3.6.1.3) was purified and incorporated into phosphatidylcholine liposomes. Freeze-fracture analysis of the reconstituted system reveals intramembrane particles formed by ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecules which are randomly distributed on concave and convex fracture faces. The reconstituted ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ performs active Na⁺,K⁺-transport. The distribution of particles as well as the rate of active transport are directly proportional to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ protein concentration used for reconstitution, while the total amount of sodium and potassium ions exchanged by ATP per volume vesicle suspension reaches maximum when each vesicle contains on the average more than two particles. ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ pretreated with ouabain or vanadate yields the same particle density and vesicle size as control enzyme. However, detergent-denatured enzyme loses its ability to form intramembrane particles or to increase the vesicle size indicating that the lipids surrounding the protein part of the molecule are essential for the reconstitution process. The vesicle diameter increases as a function of the number of particles per vesicle. Histograms of the size distribution become wider with increasing intramembrane particle density and tend to show more than one maximum.     

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets

The phospholipid requirement of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase activity}$. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

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.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

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.     

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.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

Studies on (K+ + H+)-ATPase IV. Effects of phospholipase C treatment

(1) The total phospholipid content of a gradient purified ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase and K⁺ stimulated $\text{p-}\text{nitrophenylphosphatase}$ activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP and temperature dependence of the gastric ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase, but slightly decreases the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for K⁺. (5) Phospholipase C treatment lowers the $\text{Ado}\text{PP[}\text{NH}\text{]P}$ binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

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.     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Photoconversion and regeneration of active protochlorophyll(ide) in mutants defective in the regulation of chlorophyll synthesis

Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-d¹² and $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$accumulate 2-fold, $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ 5- to 6-fold, and $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$ 15-fold more protochlorophyll(ide) than the wild type.The amount of photoconvertible protochlorophyll(ide) accumulated in darkness is the same in all genotypes, despite the large differences in total protochlorophyll(ide) content, indicating a constant number of photoconversion sites.When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and 10-min dark periods is higher in +/tig-d¹², $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$, but lower in $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$.There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll(ide) in leaves with the genotypes +/+, +/tig-o³⁴, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ are similar or identical.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg²⁺, the plasma membrane preparation exhibits a Ca²⁺-dependent ATPase activity of 2 μmol P_i per h per mg protein. It is suggested that this $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity is related to the measured Ca²⁺ transport which was characterized by $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and Ca²⁺ of $\text{44 ± 9 μ}\text{M}$ and $\text{0.25 ± 0.10 μ}\text{M}$, respectively. Phosphorylation of plasma membranes with [γ-³²P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca²⁺-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and from the catalytic subunit of $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca²⁺-pump in plasma membranes isolated from nucleated cells.