Effects of an ATP site affinity analog on some conformational and enzymatic properties of the canine kidney (Na+ + K+)-ATPase.

We have shown previously that the canine kidney Na+,K+ pump [Na+ + K+)-ATPase) reacts with the ATP affinity analog p-fluorosulfonylbenzoyladenosine (FSBA). At 20 degrees C, we find the time-course of this reaction to be that predicted for a first-order reaction accompanied by competing solvolysis of the reagent. The FSBA-inactivated (Na+ + K+)-ATPase retains the ability to move between the E1 and E2 conformations that predominate in Na+ and K+ medium, respectively. Therefore, FSBA reaction with the enzyme does not interfere significantly with either its alkali metal cation binding or its conformational freedom. The ability of ATP to influence the enzyme's conformation by binding to the high-affinity nucleotide site is decreased, however, in proportion to the degree of inhibition of enzyme activity by FSBA. In addition, the ability of the enzyme to shift from the E1 to the E2 conformation through the (ATP + Na+)-dependent phosphorylation cycle is inhibited by FSBA treatment, as shown by the decreased ability of these substrates to stimulate the K+-dependent p-nitrophenylphosphatase activity. Both of these effects are consistent with specific reaction of FSBA with the ATP binding site of the enzyme. An additional effect of FSBA treatment is that it causes loss of p-nitrophenylphosphatase activity, but to a lesser extent than (Na+ + K+)-ATPase or Na+-ATPase activity. Binding of p-nitrophenylphosphate to the enzyme is apparently unaffected by FSBA treatment, since the Km for p-nitrophenylphosphate is not changed.

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.

A widely expressed novel C2H2 zinc-finger protein with multiple consensus phosphorylation sites is conserved in mouse and man.

We have cloned a murine cDNA whose deduced sequence encodes a 455-amino-acid (aa) zinc-finger protein (Zfp), PZf (penta Zf protein). Sequence analysis shows that PZf has multiple phosphorylation consensus sites for casein kinase II and protein kinase C in its N-terminal portion. This region (aa 1 to 197), which does not share significant homology to known aa sequences, has a number of charged (39 Glu, 15 Asp, 23 Lys, 22 Arg) and hydroxyl (20 Ser, 12 Thr) aa. This potentially phosphorylatable region is followed by five C2H2 Zf in the middle of the protein. The Pzf gene is transcribed as a 5-kb mRNA in all murine tissues examined. The human genome also seems to contain one or more Pzf-related genes.

Effect of aging on intestinal absorption of aromatic amino acids in vitro in the rat.

Whole-thickness everted jejunal rings were used to measure uptake of L-tyrosine (L-Tyr), L-phenylalanine (L-Phe), and L-tryptophan (L-Trp) in 6-, 12-, and 24-mo-old rats. The rate of uptake of all three amino acids (1 mM) was significantly reduced after 20 min of incubation in 24-mo-old compared with 6-mo-old rats. Results of influx (2 min) of 0.5-40.0 mM L-Phe and L-Trp suggested an increased affinity but decreased capacity for the transporter with age; these differences were significant for L-Trp (P less than 0.05). Respective values for apparent Kt and Vmax were the following: L-Phe 6-mo-old rats, 5.46 mM and 1.03 mumol.g-1.2 min-1; 24-mo-old rats, 2.54 mM and 0.51 mumol.g-1.2 min-1; L-Trp 6-mo-old rats, 5.09 mM and 0.38 mumol.g-1.2 min-1; 24-mo-old rats, 3.20 mM and 0.25 mumol.g-1.2 min-1. Values for 12-mo-old rats fell in between the other two age groups.

5'-p-fluorosulfonylbenzoyladenosine as an ATP site affinity probe for Na+, K+-ATPase.

We have investigated the suitability of 5'-p-fluorosulfonylbenzoyladenosine (FSBA) as an ATP site affinity probe for the canine kidney Na+, K+-ATPase. The purified enzyme is slowly inactivated by this compound in suitable buffers, losing about half of its activity over a two-hour period. The rate of inactivation is more rapid in 0.1 M KCl than in 0.1 M NaCl. Low concentrations of ATP protect the enzyme against inactivation, with half-maximal effects at 4 microM ATP in 0.1 M NaCl and 350 microM ATP in 0.1 M KCl. ADP also protects against FSBA inhibition, but AMP is ineffective when present at 100 microM levels. This pattern is consistent with the previously described nucleotide specificity of the Na+, K+-ATPase. Addition of protective amounts of ATP after inactivation has occurred does not restore enzyme activity, indicating that inhibition is irreversible. Measurement of the concentration-dependence of FSBA inactivation suggests an apparent Kd for binding of this compound well above 1 mM, the solubility limit of the analog. This finding is reinforced by the failure of 1 mM FSBA to compete effectively with ATP for the high-affinity ATP site of the enzyme. Nevertheless, attachment of the analog to this site is indicated by its ability to prevent [3H]-ADP binding in proportion to the number of sites it has inactivated. Studies with [3H]-FSBA show that about 1 mole of the analog attaches specifically to the alpha subunit per mole of enzyme inactivated. A similar amount of nonspecific labeling also occurs with negligible effect on enzyme activity. These findings suggest that FSBA may be useful in probing the topography of the high-affinity ATP binding site of the Na+, K+-ATPase and related enzymes.

Carbonyl cyanide phenylhydrazones as probes of the anionic activator site of the human erythrocyte glutathione adduct transport ATPase.

We have previously shown that the ATPase activity associated with the erythrocyte glutathione adduct transporter is also stimulated by 2,4-dinitrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, both well-known anionic and lipophilic uncouplers of oxidative phosphorylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazones as activators of the ATPase. All of the compounds tested stimulated the ATPase to similar extents, based on Vmax values. The K0.5 for stimulation of the ATPase depended on the electron-withdrawing characteristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slope of this relationship, with lower K0.5 values for electron-withdrawing substituents, suggests that an anionic residue in the active site partially discourages binding of this class of activators. ortho-Substituted carbonylcyanide phenylhydrazones do not follow this relationship, but show lower apparent affinities than expected from their pKa values. This finding suggests that steric effects in that region of the binding site negatively influence the affinity.

2,4-Dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone activate the glutathione S-conjugate transport ATPase of human erythrocyte membranes.

2,4-Dinitrophenol (DNPOH) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), two classical uncouplers of mitochondrial oxidative phosphorylation, were found to stimulate human erythrocyte membrane vesicle ATPase activity. Both compounds competed with S-(2,4-dinitrophenyl)glutathione (DNPSG) for activation of the glutathione S-conjugate transport ATPase. Stimulation of the ATPase by DNPOH or FCCP occurred with Vmax values 4-6 times greater than that with DNPSG. The K0.5 for DNPOH (195 microM) was similar to that of DNPSG (196 microM), while that for FCCP (4.3 microM) was 40 times lower. Vanadate inhibits both the DNPOH- and FCCP-stimulated ATPase activities, as previously reported for the glutathione S-conjugate ATPase. The stimulation of erythrocyte vesicle ATPase activities by these classical uncoupling agents does not result from increased proton conductance across the vesicle membrane: monensin, gramicidin and nystatin, all of which increase proton conductance, but by different mechanisms, do not stimulate erythrocyte vesicle ATPase activity. Verapamil, a known P-glycoprotein ATPase activator also does not stimulate human erythrocyte membrane ATPase activity. These results show that relatively small, monoanionic lipophilic compounds such as DNPOH and FCCP can activate the glutathione S-conjugate transport ATPase. The higher Vmax values for activation by these agents than by DNPSG make possible a more sensitive assay of this transport ATPase activity. The results raise the question of whether these substances and other small anionic, lipophilic compounds are also transported by this system.

CNTF overproduction hastens onset of symptoms in motor neuron degeneration (mnd) mice.

Ciliary neurotrophic factor (CNTF) promotes the survival of motor neurons, in vitro and in vivo. Moreover, CNTF can block the degeneration of injured or diseased motor neurons in young rodents. Motor neuron degeneration (mnd) mutant mice display adult onset symptoms reflecting progressive motor debilitation and provide a model in which to test the hypothesis that CNTF can prevent the loss of these motor functions. We generated mnd mice that harbor a genomically integrated transgene, resulting in overexpression of the encoded CNTF protein in these mice. In contrast to the beneficial effects of CNTF in preventing motor neuron degeneration in other experimental paradigms, we report that overproduction of CNTF increased the rate of onset of motor disease symptoms in mnd mice and the presence of the transgene correlated with low adult body weight in mnd and wild-type genetic backgrounds.

Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway.

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. p190 inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by p190. Local regulation of p190 could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of p190 by integrin and Src, both implicated in neural plasticity.

A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury.

Within the central nervous system (CNS) ciliary neurotrophic factor (CNTF) is expressed by astrocytes where it remains stored as an intracellular protein; its release and function as an extracellular ligand are thought to occur in the event of cellular injury. We find that overexpression of CNTF in transgenic mice recapitulates the glial response to CNS lesion, as does its injection into the uninjured brain. These results demonstrate that CNTF functions as an inducer of reactive gliosis, a condition associated with a number of neurological diseases of the CNS.

[Experiences with the psychometric system combitest in the evaluation of the postnarcotic arousal period]. / Erfahrungen beim Einsatz des Psychometriesystems COMBITEST bei der Untersuchung des Aufwachverhaltens im Anschluss an Allgemeinanästhesien.

Since the introduction of new German health legislation, operations formerly performed on an in-patient basis have been transferred to the out-patient sector. Due to this development evaluating the postnarcotic arousal period has become increasingly important. Vigilance, reactivity and state of health determine a patient's well-being and time of discharge after an operation and consequently influence economical and legal issues. Former studies investigating the arousal period have mainly focused on patient interviews and assessment of patients by staff. The aim of our study was to objectivize postnarcotic vigilance and reactivity (critical flicker fusion frequency and multiple choice reaction) using neurophysiological monitoring (psychometric system Combitest 2: CFF, EWR, MWR). The applied methods have been defined as valid, reliable and sensitive tools to investigate this question. Inter-individual and gender-specific differences as well as variations due to the type of anaesthetic employed can be detected. The study participants assessed the test battery's practicability and overall described this method as easy to use. Vigilance (CFF), basic and complex sensomotor reactivity (performance in simple and multiple choice tests) were restored after a postanaesthetic phase of 90 minutes (narcotics applied: isofluran and propofol). The results show that the use of modern anaesthetics does not justify the current medical and legal claim to 6 hours of postanaesthetic observation. The computer-supported psychometric system Combitest 2 has recently been used to assess the arousal phase after application of different types of anaesthetical procedures (TIVA, volatile, balanced) and has been found to be superior to parameters traditionally used to assess discharge criteria (time until patient opens eyes, orientation concerning time and location, time spent in postanaesthetic recovery room). Besides employing our study methods, subjective factors such as well-being and pain sensation should also be evaluated when determining the time of a patient's discharge.

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton.

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.