Acid–base regulation in isolated gill cells of the goldfish (Carassius auratus)

Mechanisms of acid release and intracellular pH (pH_i) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH_i was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na⁺ channel and Na⁺/H⁺ exchanger (NHE) inhibitor, had no effect on pH_i, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH₄Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na⁺. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO₃^–-transport inhibitor, caused a slow acidification of pH_i, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A₁, an inhibitor of V-ATPase, had no effect on steady-state pH_i and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na⁺-dependent HCO₃^– transport is the dominant mechanism besides an NHE and V-ATPase to control pH_i in goldfish gill cells.Communicated by G. Heldmaier     

Chemiosmotic coupling of ion transport in the yeast vacuole: Its role in acidification inside organelles

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H⁺-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl^– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl^– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl^– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK _m for Cl^– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.     

Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O₂ ^–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O₂ ^–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10⁶ cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO₂ in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10⁶ cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP N-formyl-methionyl-leucyl-phenylalanine - BCECF-AM 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester - PMA phorbol 12-myristate 13-acetate - CGD chronic granulomatous disease - HMP hexose monophosphate - pHi intracellular pH     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S bimane S-glutathione - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP-SG S-(2,4-dinitrophenyl)glutathione - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydrazone - LTC₄ cysteinyl leukotriene - MDR multidrug transporter - MRP multidrug resistance protein - NEM-SG N-ethylmaleimide glutathione We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom     

Membrane proteins related to anion permeability of human red blood cells

Summary (³H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (³H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (³H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (³H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (³H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS 4,4-Diamino-2,2-dihydrostilbene disulfonic acid - DIDS 4,4-Diisothiocyano-2,2-stilbene disulfonic acid - (³H)DADS 4,4-Diamino-2,2-ditritiostilbene disulfonic acid - (³H)DIDS 4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Regulation of pH in the isolated perfused gills of the shore crabCarcinus maenas

Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l^-1 Na⁺), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw^-1·h^-1, a value of only 3.8% of net Na⁺ fluxes observed under comparable conditions. When 0.1 mmol·l^-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l^-1 ouabain, a specific inhibitor of Na⁺/K⁺-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l^-1 acetazolamide or 0.01 mmol·l^-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na⁺/H⁺ exchange, 0.1 mmol·l^-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l^-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl^-/HCO₃ ^- exchange and Na⁺/HCO₃ ^- cotransport, 0.5 mmol·l^-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l^-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H⁺-ATPase, 0.05 mmol·l^-1 N-ethylmaleimide and 0.1 mmol·l^-1 N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l^-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l^-1 HCO₃ ^-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO₃ ^-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na⁺/H⁺ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide (N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide) - amiloride 3,5-diamino-6-chloropyrazinoyl-guanidine - CA carbonic anhydrase - DBI dextrane-bound inhibitor thiadiazolesulphonamide - DCCD N N dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanato-stilbene-2,2-disulphonate - EIPA 5-(N-ethyl-N-isopropyl) amiloride - ethoxyzolamide 6-ethoxy-2-benzothiazole-sulphonamide - fw fresh weight - J _H ⁺ apparent proton flux - NEM N-ethylmaleimide - PD transepithelial potential difference - PEG-STZ polyethylene-glycol-thiadiazolesulphonamide - STTS 4-acetamido-4-isothiocyanatostibene 2,2-disulphonate - SW sea water - TRIS tris(hydroxymethyl)aminomethane     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Mechanism of Na+/proline symport inEscherichia coli: Reappraisal of the effect of cation binding to the Na+/proline symport carrier

Summary Recently we proposed that cytoplasmic acidification of low K⁺ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl^–-dependent K⁺ flux (K⁺Cl^– cotransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K⁺ transport on intracellular and extracellular pH (pH _i , pH _o ) varied either simultaneously or independently using the Cl^–/HCO ₃ ^– exchange inhibitor 4,4, diisothiocyanatostilbene-3,2-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K⁺ efflux and influx of Rb⁺ used as K⁺ congener were strongly activated at acid pH _i and alkaline pH _o . A small stimulation of K⁺ (Rb⁺) flux was also seen at acid pH _i in the absence of DIDS, i.e., when pH _i pH _o . Anti-L _l serum, known to inhibit K⁺Cl^– cotransport, prevented the pH _i -stimulated K⁺ (Rb⁺) fluxes. Subsequent to NEM treatment at pH 6, K⁺ (Rb⁺) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K⁺ (Rb⁺) fluxes in DIDS-treated cells with pH _o varied at constant physiologic pH _i . Anti-L _l , which inhibited NEM-stimulated K⁺ (Rb⁺) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site.     

Acetylcholinesterase molecular forms from rat and human erythrocyte membrane

Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N⁶, O²-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PK_I) decreased relative to cyclic AMP-dependent protein kinase type II (PK_II) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PK_I, PK_II and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP N⁶,O²-dibutyryladenosine-3, 5-cyclic monophosphate - cyclic AMP adenosine 3,5-cyclic monophosphate - PK_I type I cyclic AMP-dependent protein kinase - PK_II type II cyclic AMP-dependent protein kinase     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Synthesis of tritiated 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells

Summary The potent and specific inhibitor of anion permeability, 4,4-diisothicyanostilbene-2,2-disulfonic acid (DIDS) was synthesized in tritiated form ([³H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([³H]H₂DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [³H]DIDS was substantially faster than that of [³H]H₂DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [³H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×10⁶ molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate