Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the 'microsomes' (13000-80000 X g sediment) from pea stem tissue is strongly influenced by concentration of Mg2+ in the homogenization medium. The absence of Mg2+ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at 80000 X g upon addition of 5-10 mM Mg2+ (or Mn2+, Ca2+, Zn2+) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl2 the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg2+. Microsomes prepared with Mg2+ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase

The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca²⁺ and Mg²⁺ and, therefore, should be called (Ca²⁺ or Mg²⁺)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl₂ by CaCl₂ resulted in an ATPase activity of 92% of that with MgCl₂. Using calcium- and magnesium-ATP as substrates, apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 95 000 and also that of microvillus actin.     

Membrane reconstitution in chl-r mutants of Escherichia coli K 12.: VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles

Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5 % polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg²⁺ dependent, insensitive to inhibition by $\text{N,N′-}\text{dicyclohexylcarbodiimide}$ and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg²⁺ are able to incorporate the purified ATPase only in the presence of 2–6 mM Mg²⁺. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increases in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.     

A comparative study of microsomal and cytosolic S6 phosphatase activities in rat liver

Summary Spontaneous S6 phosphatase activities dephosphorylating Ser(P)-235 and Ser(P)-236 of the ribosomal protein S6 were measured and compared in microsomes and cytosol of rat liver. The substrate used, small (40S) ribosomal subunits ³²P-labelled in vitro by protein kinase A, contained phosphorylated S6 (mainly in the dephosphorylated form) and some minor phosphorylated species. The microsomal and cytosolic S6 phosphatase activities displayed a number of distinct properties. The microsomal activity, representing ca 20% of the S6 phosphatase activity in the post-mitochondrial supernatant, was mainly due to a type-1 phosphatase and dephosphorylated only S6. The remaining post-mitochondrial S6 phosphatase activity, which was fully recovered in the cytosol, and appeared to result from a combination of type-1 (43%) and type 2 (57%) phosphatases, acted on S6 as well as on the minor phosphorylated species. The microsomal activity was 50% inhibited by MgCl₂ (l0 mM) and was stimulated at least 4.3 fold by MnCl₂ (1 mM), while the cytosolic activity was inhibited only 18% by Mg²⁺ (10 mM) and was increased 2.2 fold by Mn²⁺ (1 mM). The microsomal activity was increased 10% (P < 0.06) by lower doses of insulin (25 U/Kg) and 14% (P < 0.05) by vanadate, but was not significantly (P > 0.10) affected by larger doses of insulin (100 U/kg), hepatectomy or cycloheximide. By comparison the cytosolic S6 phosphatase activity was unresponsive to insulin and vanadate, but was decreased 14% and 17% (P < 0.05) by hepatectomy and cycloheximide. It is concluded that (i) there. are clear differences between the microsomal and cytosolic S6 phosphatase activities, which may be relevant to their specific functions in the cell, and (ii) the inhibition of cytosolic S6 phosphatase activity by hepatectomy and cycloheximide may contribute to the increase in hepatic S6 phosphorylation induced by these treatments.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca²⁺ transport in this preparation were characterized and compared to those of (Ca²⁺ + Mg²⁺)-ATPase activity. The time course of Ca²⁺ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca²⁺/mg protein after 3–4 min of incubation. The rate of Ca²⁺ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca²⁺/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent $\text{K}\text{m}$ values for Mg²⁺ and Ca²⁺ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$)-ATPase. The presence of potassium was required for maximum Ca²⁺ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca²⁺ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{-}\text{ATPase}$ activity was $\text{K}{}^{\mathrm{+}}\text{>}\text{Na}{}^{\mathrm{+}}\text{=}\text{NH}{}_4{}^{\mathrm{+}}\text{>}\text{Li}{}^{\mathrm{+}}\text{.}\text{Ca}{}^{\mathrm{2+}}$ transport and ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM $\text{p-}\text{chloromercuribenzene}$ sulfonate. The results indicate that Ca²⁺ transport in adipocyte endoplasmic reticulum is mediated by a $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ and suggest an important role for endoplasmic reticulum in control of intracellular Ca²⁺ distribution.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca²⁺-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg²⁺ as the only cation) ATPase activity, with virtual elimination of the K⁺-stimulated component. Such treatment causes unmaksing of a latent Mg²⁺-dependent Ca²⁺-stimulated ATPase. Other divalent cations such as Sr²⁺, Ba²⁺, Zn²⁺ and Mn²⁺ were found ineffective as a substitute for Ca²⁺. Moreover, those divalent cations acted as inhibitors of the Ca²⁺-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 70 μM for ATP and the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ values for Mg²⁺ and Ca²⁺ are about $\text{4 · 10}{}^{\mathrm{?4}}\text{M}$ and 10^?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H⁺ transport have been discussed.     

Identity of microsomal glutathione-S-transferases

Summary Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferases were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and K_m's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.     

Properties of a rat liver smooth microsomal subfraction not aggregated by Mg2+

A smooth microsomal fraction (smooth II microsomes) was earlier isolated and characterized in a number of investigations. Using a three-layer discontinuous sucrose gradient containing Mg²⁺ this fraction was divided into two subfractions (IIa and IIb) by a single centrifugation. The smooth IIa fraction proved to be a purified smooth microsomal fraction of specific composition. It contains high amounts of cytochromes $\text{b}{}_5$ and P-450, low activities of other electron transport enzymes and glucose-6-phosphatase, and no UDP-glucuronic acid transferase. No membrane or enzyme synthesis is induced in this subfraction by treatment with phenobarbital or methylcholanthrene. It appears that the membranes of smooth IIa microsomes derive from the smooth endoplasmic reticulum and are devoted to specific functions.     

Inhibition of (Na+ + K+)-ATPase by ouabain: Involvement of calcium and membrane proteins

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ Mg²⁺-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000–34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca²⁺. The recovery of the original resistance to ouabain required specifically Ca²⁺ and was associated with a binding of the solubilized proteins to the membrane.     

Effects of lanthanum on calcium-dependent phenomena in human red cells

Lanthanum (0.25 mM) does not penetrate into fresh or Mg²⁺-depleted cells, whereas it does into ATP-depleted or $\text{ATP + 2,3-diphosphoglycerate-depleted cells}$, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca²⁺-efflux completely and inhibits $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg²⁺-depleted cells Ca²⁺-Ca²⁺ exchange is inhibited by lanthanum. Ca²⁺-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca²⁺-leak ± S.D. amounts to $\text{0.28 ± 0.08 μ}\text{mol/l}$ of cells per min, whereas in KCl medium to $\text{0.15 ± 0.04 μ}\text{mol/l}$ of cells per min at 2.5 mM [Ca²⁺]_e and 0.25 mM [La³⁺]_e pH 7.1.Lanthanum inhibits Ca²⁺-dependent rapid K⁺ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K⁺ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca²⁺-loaded cells, however, is blocked by lanthanum owing to Ca²⁺-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca²⁺ concentrations.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.