Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca²⁺ resulted in inactivation of the Ca²⁺-stimulated ATPase activity. Omission of Ca²⁺ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca²⁺-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca²⁺-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca²⁺-stimulated, Mg²⁺-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg²⁺-ATPase slightly. CCCP was much more specific for the Ca²⁺-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca²⁺ for inactivation was also quite specific.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

K+-valinomycin and chloride conductance of the human red cell membrane. Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone

The major form of microsomal cytochrome P-450 induced by trans-stilbene oxide in the liver of male Sprague-Dawley rats was purified and characterized, and compared with the isolated cytochrome P-450 B2 forms from phenobarbital- and 3-methylcholanthrene-pretreated animals. The apparent subunit molecular weight of the trans-stilbene oxide-induced cytochrome was found to be 53 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absorbance maximum of the carbon monoxide complex of the ferrous cytochrome was 450 nm. Reconstitution of the N-demethylase activity towards three different substrates showed high and similar activities with the cytochrome P-450 B2 forms from trans-stilbene oxide or phenobarbital-treated rats, with one exception. Amino-acid analysis also showed a very high degree of similarity between these two forms. Upon proteinase treatment with three different proteinases the trans-stilbene oxide-induced cytochrome demonstrated in each case a peptide pattern identical to that obtained with the phenobarbital-induced B2 form. Furthermore, both forms are completely immunologically cross-reactive. We therefore conclude from these experiments that the liver microsomal P-450 B2 from trans-stilbene oxide and phenobarbital-treated rats are very closely related, if not identical.     

Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone

Experiments were performed under several conditions seeking evidence for turnover of protein-bound lipoic acid in Escherichia coli analogous to that described for the 4′-phosphopantetheine moiety of the E. coli acyl carrier protein. Pulse-chase, chase experiments using both low and saturating concentrations of lipoic acid, chase experiments in the presence of chloramphenicol, which prevents incorporation of lipoic acid into the protein-bound form, and chase experiments in cells in which the free pool of lipoic acid was reduced by osmotic shock all failed to demonstrate any turnover of protein-bound lipoic acid.     

Significant enhancement of photobiological H2 evolution by carbonylcyanide m-chlorophenylhydrazone in the marine green alga Platymonas subcordiformis

A marine green microalga, Platymonas subcordiformis, photo-synthetically generates H(2) but only transiently at a negligible yield when exposed to light after a period of dark anaerobic incubation. A protonophore uncoupler, carbonyl cyanide m-chlorophenylhrazone (CCCP) significantly increased the yield of H(2) photo-production. CCCP optimally at 15 microM gave 4.9 ml H(2) after 8 h light irradiation in 1 l algal cell culture at 1.8 x 10(6) cells ml(-1). The H(2) yield at 15 microM CCCP was increased by 240-fold when compared to the control. This improvement may be by CCCP disrupting the proton motive force thus facilitating proton transfer across the thylakoidal membrane.     

Effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli halotolerance.

The growth-inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was less on members of the family Enterobacteriaceae (halotolerant organisms) than it was on species of Vibrio (moderately halophilic organisms). When sodium chloride concentration increased from 0.5 to 0.85 M, this effect was more pronounced for Escherichia coli; it remained relatively stable for Vibrio spp. The effect of carbonyl cyanide m-chlorophenylhydrazone was antagonized by the addition of glycine betaine or proline or by growth in a rich medium.     

Effect of alpha-actinin on actin structure. Actin ATPase activity

The accumulation of low molecular weight RNAs in Escherichia coli cells following amino acid or energy source starvation was examined using two-dimensional polyacrylamide gel electrophoresis. 32P-labeled small RNA prepared from serine- or isoleucine-starved stringent strain (relA+) cells was shown to display gel patterns that were grossly different from that of unstarved cells. It appears that the deprivation of serine or isoleucine has little or no inhibitory effect on the accumulation of transfer RNA cognate to the deprived amino acid. This is demonstrated by a relative increase in the concentrations of small RNAs that can be charged with serine or isoleucine following starvation of these amino acids. However, small RNAs labeled during starvation of phenylalanine or energy source showed gel patterns similar to that of control cells. This suggested a heterogenous response in the accumulation of some low molecular weight RNAs, presumably transfer RNAs, following starvation of different amino acids.     

Effect of medium viscosity on the activity of myosin ATPase

The influence of increased medium viscosity on the activity of myosin Ca2+-ATPase has been studied in 28, 45 and 60% water-sucrose solutions at 25 degrees C. In the wide range of viscosities (10 divided by 430 mp) the rate constant of ATP hydrolysis displays the negative power-law dependence on solution viscosity with an index approximately -0,5. The obtained data confirm an idea about the existence of direct connection between the low-frequency liquid relaxations and structural dynamics of proteins and enzymes.     

Effect of physico-chemical factors on anion-sensitive ATPase activity

Effects of temperature, pH and anions on the ATPase activity of submitochondrial particles of rat liver, rat heart, mouse liver, of red blood cell membranes, and of soluble enzyme of rat liver, mouse liver mitochondria were studied. The temperature relationships of membrane-bound and soluble ATPases have the breaks at 18-21 degrees C and 30-32 degrees C. These breaks were not shifted by sulfite, thiocyanate, methanol, glycerol and GTP. The pH changes from 6.0 to 8.5 produced no effect on the temperature relationships of ATPase activities but, strongly influenced the rate of ATPase reaction. The conformity between the obtained data and earlier proposed mechanism of anion control over anion-sensitive ATPase activity was discussed.     

Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.     

Effect of anions on the ATPase activity of submitochondrial particles

The effects of anions on the ATPase activity of submitochondrial particles from mouse liver cells were investigated. Thiocyanite decreased the ATP hydrolysis, acting as a competitive inhibitor with respect to sulfite. All the anions tested changed the ATPase activity noncompetitively towards Mg-ATP. The hydrolysis of CTP, GTP, ITP and UTP was insensitive to sulfite and thiocyanate. In the presence of Mn2+, Ca2+, Co2+, Zn2+ and Ba2+ an anion-dependent hydrolysis of ATP took place. It was assumed that the anions control the rate of the limiting step of the ATPase reaction, since sulfite and thiocyanate change the activation energy of ATP hydrolysis. The data obtained are discussed in terms of a previously proposed mechanism of the anions effect on the activity of mitochondrial ATPase.     

Effect of ultrasound on the ATPase activity of brain microsomes

Ultrasonic effects (0.88 MHz, 0.8 Wt X cm-2) on Na+, K+-ATPase and Mg2+-ATPase changed the values KM, Vmax, delta H not equal to, delta S not equal to and the activation energy, which can point to some changes in the structure of lipoprotein enzyme complexes.     

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 h of recovery period after the depletion of LPL with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.     

Effect of tetraphenylboron on the calcium-dependent ATPase activity of sarcoplasmic reticulum

The apparent effect of tetraphenylboron on the Ca2+-dependent ATPase activity is rather complex being dependent of the physical state of the membrane. In leaky vesicles, tetraphenylboron decreases the affinity of the enzyme for Ca2+, and high concentrations (100 microM) inhibit the enzyme. These effects are mediated by the membrane structure. In native vesicles, tetraphenylboron induces an apparent stimulation of the maximum hydrolytic activity and a decrease of the Ca2+ gradient formed due to specific Ca2+ releasing activity of the mentioned drug.