Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N-Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation-dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.     

A study of the control of NADP(+)-dependent isocitrate dehydrogenase activity during gonadotropin-induced development of the rat ovary

The concentration of cytoplasmic NADP(+)-dependent isocitrate dehydrogenase increased 20.2-fold during gonadotropin-induced development of the immature rat ovary. Measurement was by protein (Western) blotting using polyclonal antibodies raised against purified enzyme from the porcine corpus luteum. The increase in enzyme concentration during development correlated well with the 18.5-fold increase observed for the specific activity of the enzyme in the cytosolic fraction. An immunochemical similarity was demonstrated between the cytoplasmic enzyme from the ovary, testes, placenta, skeletal muscle, brain, liver, kidney, mammary and adrenal gland. However the mitochondrial NADP(+)-dependent isocitrate dehydrogenase from these tissues was found to be immunochemically distinct from the cytoplasmic enzyme. The concentration of the substrate D(+/-)-threo-isocitrate in the ovaries was measured by fluorometry and found to increase 3.1-fold during hormone-induced development. The intracellular concentration of substrate was estimated to be of the same order of magnitude as the enzyme concentration. We conclude that the increase in cytoplasmic NADP(+)-dependent isocitrate dehydrogenase activity observed during the gonadotropin-stimulated development of the rat ovary is due to increased concentration of enzyme rather than to an activation of the enzyme. The activity of the enzyme in vivo appears to be regulated by the availability of the substrate D(+/-)-threo-isocitrate.     

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis

The H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca²⁺ and a number of other divalent cations (Co²⁺, Mn²⁺, Zn²⁺). By contrast, the H⁺-translocating ATPase (H⁺-ATPase) located on the same membrane is insensitive to Ca²⁺. Here we examine the mechanism and feasibility of regulation of the vacuolar H⁺-PPase by cytosolic free Ca²⁺ under the conditions thought to prevail in vivo with respect to Mg²⁺, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca²⁺ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg₂PPi) at one site, inhibitory binding of Mg₂PPi to a lower-affinity second site, binding of activator (Mg²⁺) at a third site, and direct binding of Ca²⁺ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca²⁺ or CaPPi are explicable only if Ca²⁺, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca²⁺ on the substrate concentration dependence of H⁺-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H⁺-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca²⁺ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

The disulfide bonded ring of iso-rANP, unlike that of rANP, has potent cardiovascular activity

Iso-atrial natriuretic peptide (iso-rANP), a 45 amino acid peptide with a disulfide bond between residues 23 and 39, is a newly discovered second atrial hormone with considerable homology with rat atrial natriuretic peptide (rANP). We have reported that iso-rANP(1-45) has effects similar to rANP in anesthetized rats in causing hypotension, a decrease in heart rate, and an increase in urine flow and electrolyte excretion. In the present experiments, we found that, unlike the ring peptide of rANP (rANP(105-121)), bolus injections of the ring peptide of iso-rANP (iso-rANP(23-39)) had considerable effects on the circulation but only small effects on the kidney, compared with iso-rANP(1-45). However, the effects of iso-rANP(23-39) in causing hypotension and decreasing heart rate were transient compared with those of iso-rANP(1-45). When we substituted a glycine for arginine into the ring portion of iso-rANP at position 36, so that there were only three amino acids different from the ring of rANP, biological activity of iso-rANP(23-39) was retained. In our bioassay system, all of the circulatory effects and most of the renal effects of rANP are mediated via vagal sensory afferents. We found that the effects of the ring peptide of iso-rANP on the circulation and the kidney were unchanged following vagotomy. The results indicate that iso-rANP(23-39) mediates at least some biological effects by membrane receptor mechanisms different from those of rANP and that the ring portion of iso-rANP probably contributes to nonvagally mediated responses of iso-rANP(1-45).     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg²⁺, and at pH 7.4 in the presence of Mg²⁺. Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.     

Stoichiometry of a half-turnover of band 3, the chloride transport protein of human erythrocytes

The kinetics of human red blood cell Cl transport have been studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4'-dinitro-2,2'-stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per ghost, which is abolished by pretreatment with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), appears to represent the Cl that is transported during the first half-turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.