Adaptations of sodium balance to low pH in a sunfish (Enneacanthus obesus) from naturally acidic waters

Summary Net sodium flux (J _net), sodium influx (J _in), and sodium efflux (J _out) were measured in two sunfish,Enneacanthus obesus (acid-tolerant) andLepomis gibbosus (less acid-tolerant), during 24 h exposure to soft water of pH's 4.0 and 3.5.E. obesus exhibited a mild transitory disturbance at both pH's caused by inhibitedJ _in and slightly stimulatedJ _out. Body and plasma ion concentrations ofE. obesus were measured weekly during exposures for 5 weeks to acidified artificial soft water (ASW). Body sodium concentration declined 30% during 2 weeks exposure to pH 3.5 but no further during the next three weeks. Exposure to pH 4.0 had no effect on body sodium concentration during the entire 5 weeks. Plasma sodium concentration declined 15% over a 3 week period at pH 3.5; there was no further change in the next two weeks. Plasma potassium concentrations, which were measured after 4 and 5 weeks at pH's 5.8 and 3.5 in ASW, were not significantly different. In a separate two week long experiment, plasma sodium concentration ofE. obesus in ASW was correlated with pH between pH's 3.5 and 7.5. This effect was mainly due to increases above pretreatment levels at pH 4.5 and above. Increased ambient sodium and calcium concentrations had no effect on body sodium concentration ofE. obesus at pH 5.8, but mitigated the effects of exposure to pH 3.5. Increased calcium concentrations up to 25 M at pH 3.5 increased body sodium concentration, but higher concentrations had no additional effect. Body potassium concentration and body water concentration ofE. obesus were linearly related to body sodium concentration under a wide variety of external conditions. This suggests the presence of a mechanism by whichE. obesus regulates plasma sodium levels and body fluid compartments in response to sodium loss. In contrast toE. obesus, L. gibbosus showed larger sodium losses at low pH resulting from greater acceleration ofJ _out; those exposed at pH 3.5 died in less than 12 h.L. gibbosus also had reduced body and plasma sodium concentrations at pH 4.5 and below; those at pH 4.0 were the lowest. Body potassium concentration ofL. gibbosus was reduced in those fish exposed to pH 4.0 and below, but body water was increased. Thus there are striking differences in the ability to regulate ion and water balance at low pH between an acid-tolerant specialist (E. obesus) and a less acid-tolerant generalist (L. gibbosus).Abbreviations ASW artificial soft water - WBM wet body mass - DBM dry body mass     

The effect of food consumption on sodium and water balance in the predaceous diving beetle,Dytiscus verticalis

Summary Sodium and water balance ofDytiscus verticalis in fresh water were investigated under three feeding regimes: unfed, and fed a diet either low or high in sodium chloride. Unfed sodium influx was 0.13 and sodium efflux was 0.74 moles/100 gwm·h. These values are low in comparison with most freshwater animals. The electrical potential difference across the integument in artificial soft water (ASW) was about 150 mV smaller than the potential necessary to maintain sodium balance in the absence of active transport. However, sodium influx did not show saturation kinetics over an external concentration range of 91 to 1725 M. Unfed beetles failed to arrest net sodium loss to baths that were initially distilled water or ASW, even when bath sodium concentrations reached 75–298 M. The long-term rate of net sodium loss ranged from 0.61 to 4.4 moles/100 gwm·h for four sets of animals. Beetles decreased sodium efflux during a period of fasting. During subsequent feeding, beetles fed a high sodium diet (HSD) increased sodium efflux while beetles fed a low sodium diet (LSD) maintained low rates of sodium efflux. HSD fed beetles increased body sodium and hemolymph sodium concentration, and expanded extracellular fluid, relative to LSD fed beetles. Thus beetles cannot achieve sodium balance in fresh water without dietary sodium input, although they are able to regulate sodium loss.Abbreviations gwm grams wet mass - ASW artificial soft water - DW distilled water - HSD high sodium diet - LSD low sodium diet - ECF extracellular fluid volume     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Comparison between (RS)-nipecotic acid and GABA transport in cultured astrocytes: Coupling with two sodium ions

The sodium ion dependency of the uptake of (RS)-nipecotic acid into astrocytes in primary cultures has been studied by performing kinetic analysis at different sodium ion concentrations (16–151 mM).V _max of the saturable component of the astroglial (RS-nipecotic acid uptake is clearly affected by the sodium ion concentration whereasK _m surprisingly remains unaffected. At high (RS)-nipecotic acid concentrations (50 M), uptake rates as a function of the sodium ion concentration were clearly sigmoid. This sigmoid shape was not obvious at lower concentrations of (RS)-nipecotic acid. The calculated Hill coefficients corresponding to all (RS)-nipecotic acid concentrations studied were approximately two. From these results it is concluded that (RS)-nipecotic acid uptake into astrocytes in primary cultures, like astroglical GABA uptake, requires the binding of at least two sodium ions per (RS)-nipecotic acid molecule transported.     

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.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Effects of six trace metals on calcium fluxes in brown trout (Salmo trutta L.) in soft water

Summary Calcium fluxes were measured simultaneously in brown trout fry maintained in an artificial soft water medium of [Ca] 20 mol·l^-1 and pH 5.6, and exposed to each of six trace metals (Al, Cu, Fe, Ni, Pb, and Zn). The trace metal concentrations represented typical and maximum levels found in acid waters experiencing declining fishery status. In the absence of trace metals, evidence is presented which suggests that ca. 91% of Ca taken up from the external medium was by extraintestinal active transport. Calcium efflux was stimulated by both concentrations of Al, Cu, Fe, and Pb. Efflux was also stimulated by [Ni] 170 nmol·l^-1 and [Zn] 3000 nmol·l^-1. In some cases, response to increased efflux was stimulation of influx. Lack of stimulation of influx resulted in negative net Ca fluxes. Net Ca losses were recorded at both concentrations of Al, Pb, and Ni, lower concentrations only of Fe, and higher concentrations only of Cu and Zn.Abbreviations J _in influx - J _net net flux - J _out efflux Henceforward in this paper, chemical elements are referred to by their chemical symbols rather than by full names     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Microhabitat selection by tadpoles of <Emphasis Type="Italic">Buergeria japonica</Emphasis> inhabiting the coastal area

Microhabitat selection is particularly important to increase the survival rate and reproductive success of animals inhabiting heterogeneous environments. I investigated microhabitat selection of Buergeria japonica tadpoles inhabiting the stream in a coastal area to reveal how animals select their appropriate habitat under heterogeneous and unstable environments on the subtropical Okinawa Island of Japan. Tadpoles are sensitive to subtle environmental changes, and the mouths of streams in coastal areas that have intense environmental fluctuations such as desiccation and sudden changes in current velocity would be risky habitat for tadpoles. Tadpoles of B. japonica can inhabit both lotic and lentic systems. Field observations showed that, among six physical factors (water depth, water temperature, salinity, pH, current velocity, and substrate), current velocity and water temperature were key factors in microhabitat selection by tadpoles. It is likely that tadpoles stay at low current velocity sites to reduce the probability of being washed out to the sea by a sudden squall and that selection of warmer sites would accelerate development of tadpoles so as to escape the heterogeneous aquatic habitat sooner.     

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

Summary In jejunal brush-border membrane vesicles, an outwardly 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 indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater 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 stepwise 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±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 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. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _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 divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects 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.     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

Catalytic properties of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> KM-1 phenylalanine ammonia-lyase

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K _M 1.75 ± 0.44 mM and V _max 3.01 ± 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K _i 3.38 ± 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57°C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.     

Acetylcholinesterase from desert Cobra (Walterinnesia aegyptia) venom: Optimization and kinetics study

Acetylcholinesterase (AChE) was investigated inWalterinnesia aegyptia venom and characterized with respect to its kinetic properties. It was found that 4.0 ug of crude venom protein and an incubation time of 4.0 min were suitable conditions for linearity of AChE activity at 25°C. The optimum strength of the sodium phosphate buffer was 0.05 M, and the optimum pH was 7.75. The optimum temperature was 30°C. The activation energy and the heat of activation were observed to be 6510 and 5922 cal/mole. The AChE was specific for acetylthiocholine but it did not hydrolyse butyrylthiocholine. The optimum substrate concentration was 3.0 mM but at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (K_m, V_max, k_cat, and k_sp) were established at each order of the reaction.Abbreviations AChE acetylcholinesterase - ASCh acetylthiocholine - K_m Michaelis-Menten constant - V_max the limiting maximal velocity - AChE_a acylated enzyme - k_cat turnover number - k_sp specificity constant     

“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.     

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum

Summary A quantitative histochemical method to determine the apparent Km and V _max values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025–1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at 550 nm. The ratio between the absorbance at 550 and 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 m both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26±0.081 mM (weighted regression analysis) and 0.28–0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme.The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 m, incubation time 3 min, 37°C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data ±SD from the jejunal villi read as follows. Apical: Km=0.81±0.438 mM, V _max=3.99±1.217 absorbance units (A) and Km=0.87±0.428 mM, V _max=4.02±1.191 A, respectively. Basal: Km=0.82±0.261 mM, V _max=3.26±0.719 A and Km=0.77±0.184 mM, V _max=3.04±0.518 AU, respectively. As demonstrated by factorial analysis of variance only V _max is influenced by the villus position.Supported by the Deutsche Forschungsgemeinschaft GU 184/1     

Net uptake of potassium in Neurospora. Exchange for sodium and hydrogen ions

Net uptake of potassium by low K, high Na cells of Neurospora at pH 5.8 is accompanied by net extrusion of sodium and hydrogen ions. The amount of potassium taken up by the cells is matched by the sum of sodium and hydrogen ions lost, under a variety of conditions: prolonged preincubation, partial respiratory inhibition (DNP), and lowered [K]_o. All three fluxes are exponential with time and obey Michaelis kinetics as functions of [K]_o. The V _max for net potassium uptake, 22.7 mmoles/kg cell water/min, is very close to that for K/K exchange reported previously (20 mmoles/kg cell water/min). However, the apparent K_m for net potassium uptake, 11.8 mM [K]_o, is an order of magnitude larger than the value (1 mM) for K/K exchange. It is suggested that a single transport system handles both net K uptake and K/K exchange, but that the affinity of the external site for potassium is influenced by the species of ion being extruded.     

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils.     

KINETICS OF THE SODIUM-DEPENDENT TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

The kinetics of transport of gamma-aminobutyric acid [2,3-³H] by synaptosomes from rat brain was studied by means of a rapid filtration technique. The rate of uptake was proportional to the protein concentration over the range 0.05—0.2 mg of synaptosomal protein per ml. Although apparent allosteric kinetics were observed with sodium, transport followed simple saturation kinetics with respect to GABA and no heterotropic, cooperative effects of GABA on sodium on kinetics were observed. A minimum of three interacting sodium sites is suggested the basis of Hill plots of the sodium data. Both the apparent K_m and V_max for GABA were functions of the sodium ion concentration but the effect of sodium was considerably greater on V_max than on the apparent K_m The V_max for GABA was 1.1 ± 0.5 nmol.min^?1 mg^?1 of protein at 95 mm sodium and decreased to 12 per Cent of this value at 19 mm sodium. The apparent K^m for GABA increased from 4.0 ± 1.0 μm at 95 mm sodium to 8.4 ± 2.0 μm at 19 mm sodium. Potassium was a noncompetitive inhibitor with respect to GABA and did not affect the apparent cooperativity observed with sodium. These findings are discussed in terms of models of GABA transport.