Evidence for disaggregation of oligomeric G(o)alpha induced by guanosine-5;-3-O-(thio)triphosphate activation

Myristoylated G_o was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G_o) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G_o, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5"-3-O-(thio)triphosphate (GTPS) activation induced disaggregation of the G_o oligomers to monomers. The G_o was crosslinked by a cross-linker, N,N"-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G_o was pretreated with GTPS. Immunoblot analysis also demonstrated oligomerization of the purified G_o proteins and its disaggregation triggered by GTPS. These results provided direct evidence for the disaggregation–coupling theory and the disaggregation action of GTPS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

A note on the kinetics of uptake of d-glucose by the food yeast,Candida utilis

Unlike other yeasts so far investigated, the d-glucose carrier of Candida utilis (strain NCYC 737) appears to change affinity for d-glucose according to its exogenous concentration. When the concentration of d-glucose was <0.4 mM, the apparent K _m 0.2 mM; at >0.4 mM, the K _m 10 mM.     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

Do neurons have a voltage or a current threshold for action potential initiation?

The majority of neural network models consider the output of single neurons to be a continuous, positive, and saturating firing ratef(t), while a minority treat neuronal output as a series of delta pulses (t — t _i ). We here argue that the issue of the proper output representation relates to the biophysics of the cells in question and, in particular, to whether initiation of somatic action potentials occurs when a certain thresholdvoltage or a thresholdcurrent is exceeded. We approach this issue using numerical simulations of the electrical behavior of a layer 5 pyramidal cell from cat visual cortex. The dendritic tree is passive while the cell body includes eight voltage- and calcium-dependent membrane conductances.We compute both the steady-state (I ^static (V _m )) and the instantaneous (I _o (V_m)) I–V relationships and argue that the amplitude of the local maximum inI ^static (V _m ) corresponds to the current thresholdI _th for sustained inputs, while the location of the middle zero-crossing ofI _o corresponds to a fixed voltage thresholdV _th for rapid inputs. We confirm this using numerical simulations: for rapid synaptic inputs, spikes are initiated if the somatic potential exceedsV _th, while for slowly varying inputI _th must be exceeded. Due to the presence of the large dendritic tree, no charge thresholdQ _th exists for physiological input.Introducing the temporal average of the somatic membrane potential (V _m) while the cell is spiking repetitively, allows us to define a dynamic I-V relationship ^dynamic ((V _m)). We find an exponential relationship between (V _m) and the net current sunk by the somatic membrane during spiking (diode-like behavior). The slope ofI/dynamic((V _m)) allows us to define a dynamic input conductance and a time constant that characterizes how rapidly the cell changes its output firing frequency in response to a change in its input.     

Direct evidence for opposite effects of D-glucose and D-glyceraldehyde on cytoplasmic pH of mouse pancreatic β-cells

The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH ₄ ⁺ and high concentrations of K⁺ on cytoplasmic pH (pH _i ) were investigated in dispersed and cultured pancreatic -cells fromob/ob mice. The cytoplasmic pH was measured with the fluorescent H⁺-indicator quene 1. The average pH _i value in resting -cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pH _i with 0.05 units. Both glibenclamide and high concentrations of K⁺ decreased pH _i . The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca²⁺ can be involved in the regulation of pH _i . In contrast to D-glucose, 10mM of D-glyceraldehyde decreased pH _i by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pH _i in pancreatic -cells.     

Involvement of cytoplasmic pH in the production of ethylene,a potent inducer of sexual development inDictyostelium mucoroides

Summary Dictyostelium mucoroides-7 (Dm 7) and a mutant MF 1 derived from it exhibit two developmental pathways: sorocarp formation occurs during the asexual process, and macrocyst formation during the sexual cycle. The two developmental pathways are mainly regulated by two chemical substances: 3,5-cyclic adenosine monophosphate (cAMP) and ethylene. Recently, we have demonstrated that cytoplasmic pH (pH_i) has a critical role for the choice of developmental pathways, higher pH_i being favourable to macrocyst formation. Thereupon, attention was riveted to the relation of pH_i to biosynthesis of cAMP and ethylene. Effect of pH_i on the production and release of ethylene, a potent inducer of macrocyst formation, was examined, using the two facing culture method. The result showed that lowered pH_i inhibits ethylene production, thus resulting in a failure of cells to form macrocysts. The accumulation of cAMP, an inhibitor of macrocyst formation, was found to vary depending on extracellular pH (pH_o), but diethylstilbestrol (DES) that is a proton pump inhibitor and also an inhibitor of macrocyst formation had no significant effect on the accumulation. Taken together these results indicate that higher pH_i may induce macrocyst formation through enhancement of ethylene production rather than inhibition of cAMP synthesis.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - pH_i cytoplasmic pH - pH_o extracellular pH - ACC 1-1-aminocyclopropane-1-carboxylic acid     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca²⁺-dependent gating of a voltage-sensitive Ca²⁺ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca²⁺ Sr²⁺ Ba²⁺ > Mg²⁺ K⁺ which is inversely related to the size of the unitary conductances K⁺ Mg²⁺ Ba²⁺ > Sr²⁺ Ca²⁺, suggesting that ion movement is not independent. In the presence of Ca²⁺, the unitary K⁺ current is reduced in a concentration- and voltage-dependent manner by Ca²⁺ binding at a high affinity site (K _0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca²⁺ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca²⁺ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca²⁺ at –41 mV predicts that two Ca²⁺ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529).     

PO2 and pH changes in the retina of the crab Ocypode ryderi: evidence for aerobic glycolysis

Summary The isolated retina of the terrestrial crab Ocypode ryderi exhibits a pronounced lactate production in spite of being supplied with sufficient O₂ (140 torr). To determine whether this lactate production is caused by hypoxic areas in the tissue or represents aerobic glycolysis, oxygen partial pressure and pH measurements with two-channel glass microelectrodes and additional biochemical analyses were carried out on this organ. Distinct profiles were obtained for O₂ partial pressure and pH inside the tissue. At a depth of 200 m different O₂ partial pressure levels could be observed depending on the O₂ partial pressure in the medium (85 torr at 280 torr and 36 torr at 130 torr, respectively). The extracellular pH displays a similar pattern; it reaches a stable value of 7.15 at 100 m inside the tissue. Lowering bath O₂ partial pressure from 280 torr to about 15 torr (hypoxia) induces a decrease of the O₂ partial pressure in the tissue with different time-courses for different tissue depths. However, hypoxia did not change the extracellular pH. Addition of antimycin A (100 mol · 1^-1) to the medium abolishes the O₂ partial pressure gradient and the delayed recovery of the tissue O₂ partial pressure after hypoxia. These results and the biochemical data suggest that in the crab retina a high glycolytic activity occurs simultaneously with oxydative carbohydrate degradation (aerobic glycolysis).Abbreviations AEC Atkinson energy charge - DC bioelectric potential - dw dry weight - HEPES N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] - PCO₂ carbon dioxide partial pressure - PO₂ oxygen partial pressure - P _tO₂ oxygen partial pressure inside the tissue - P _mO₂ oxygen partial pressure in the medium - pH_t pH inside the tissue - pH_m pH in the superfusion medium     

Kinetic study on the β-glucosidase-catalysed reaction of Trichoderma viride cellulase

A kinetic study of the -glucosidase-catalysed reaction of a commercial cellulase preparation from Trichoderma viride is described. The K _m and V _max values of the -glucosidase system were: (a) 0.5 mm and 6.6 mol/min, respectively, using p-nitrophenyl -d-glucopyranoside (pNPG) as substrate; and (b) 2.5 mm and 8.1 mol/min, respectively, using cellobiose as subtrate. The glucose effect on initial reaction velocity agrees with a mixed-inhibition pattern. The inhibition constant (K _i) values were, 0.53 and 0.39 mm with nNPG and cellobiose as substrates, respectively. The temperature and pH optima were determined. Correspondence to: A. Romeu     

“Exchange diffusion”: Rate equations for the influx of α-aminoisobutyric acid into mouse cerebrum slices containing this amino acid

Rate equations for the gross influx of -aminoisobutyric acid (AIB) into mouse cerebrum slices containing AIB have a first-order term for unsaturable concentrative influx, identical to the corresponding term for unloaded slices, and a modified Michaelis-Menten term,V_max/(1+K _t /S), for saturable concentrative influx. [V_max v _L (1+K _t /S), wherev _L =saturable component of influx,S=AIB concentration in medium, andK _t =Michaelis constant for unloaded slices.] Below a tissue AIB (T) of 19 µmol/g final wet weight,V_max increases linearly followingV_max=V ₁+m ₁ T; above that value,V _max is virtually constant. The transition is sharp. This equation is consistent with a carrier model for active transport. At the transition, intracellular AIB is about 1 molecule for every 70 amino acid residues of tissue protein, vastly more than could be accommodated by AIB-binding sites in cell membranes. The transition may come from a slow process that does not fill all sites when the tissue AIB is below the transition concentration, or from an AIB-induced phase transition in the membrane.Nomenclature AIB -aminoisobutyric acid - A radioactivity of reference; unspecified amino acid - C counts in tissue sample; carrier for transport - C _i carrier in form that reacts with intracellular substrate - C _o carrier in form that reacts with extracellular substrate - C _R counts in reference - CS complex of substrate with carrier - (CS) _i complex of substrate with carrier in formC _i - (CS) _o complex of substrate with carrier in formC _o - G counts per gram of tissue - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - k _u rate constant for first-order unsaturable uptake - K,K ,K ,K ,K _d adjustable parameters in Eqs. (9)–(13) for v, analogous to the Michaelis constant - K _d dissociation constant - K _t Michaelis constant for saturable uptake - K _t Michaelis constant for gross saturable uptake by tissue containing substrate - m ₁,m ₂ slope in Eq. (5) or (6) expressing dependence ofV_max onT orT _i ^w in Region 1 or 2 - M binding site for amino acid A - n number of data points - P number of parameters to be determined; parameter in Stein's (1981) equation, Eq. (17) in this paper - P ₁,P ₂,P ₁₂ property of tissue with unoccupied binding sites, property of tissue with occupied binding sites, property of tissue with both unoccupied and occupied binding sites, respectively - Q parameter in Stein's (1981) equation, Eq. (17) in this paper - r Pearson's correlation coefficient - Relative error RE =100{[(observed quantity – calculated quantity)/calculated quantity]²/(n –P)}^1/2 - S concentration of substrate in medium; transport substrate - S _i intracellular transport substrate - S _int AIB in medium corresponding to intracellular AIB at intersection - S _o extracellular transport substrate - T observed concentration of substrate in tissue including substrate in extracellular space and adherent fluids - T _i intracellular concentration of substrate - T _int tissue AIB corresponding to intracellular AIB at intersection - T _i ^w ,T _i ^/30 intracellular concentration of substrate withw% (30%) extracellular and adherent fluids - U observed uptake of labeled substrate by incubated tissue including substrate in extracellular and adherent fluids - U _R observed uptake of labeled substrate referred to concentration of substrate in medium - U _max adjustable parameter in Eqs. (9)–(15) for v, analogous to the Michaelis-Menten maximum rate,V _max - v influx of substrate - v _L gross influx of substrate into tissue containing substrate - v _L contribution of saturable component to gross influx into tissue containing substrate - v incremental influx, that is, gross influx into tissue that contains substrate minus influx under the same conditions into tissue that does not contain substrate - V ₁,V ₂ intercept in Eq. (5) or (6) expressing dependence ofV_max onT orT _i ^w in Region 1 or 2, respectively - V _max maximum rate in Michaelis-Menten equation - V_max apparent maximum rate defined byV_maxv_max(1+K _t /S) - V_{max 1},V_{max 2} apparent maximum rate in Region 1 or 2, respectively - V_int apparent maximum rate at intersection defining boundary between Regions 1 and 2 - w weight of incubated tissue - W _d dry weight of tissue expressed as fraction by weight - W _e extracellular and surface space of incubated tissue expressed as percent by weight - , , adjustable parameters in modified expressions for gross unsaturable influx into tissue containing substrate - , , , exponents ofS orT in Eqs. (9)–(13) for v - parameter in Stein's (1981) equation, Eq. (17), corresponding more or less tom ₁ For my wife, Lynn.     

Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase

Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Gal1,3(Fuc1,4)GlcNAc, on some N-glycans. By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian 1,3/4-fucosyltransferases. The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (K_m 16 M) to lacto-N-tetraose (Gal1,3GlcNAc1,3Gal1,4Glc; K_m 80 M) as well as to 1,3- and 1,4-galactosylated N-glycans. It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato.     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.