PURIFICATION AND PHYSICOKINETIC CHARACTERIZATION OF A GLUCONOLACTONE INHIBITION-INSENSITIVE β-GLUCOSIDASE FROM Andrographis paniculata NEES. LEAF

A gluconolactone inhibition-insensitive β-glucosidase from Andrographis paniculata (Acanthaceae) leaves has been isolated, homogeneity purified, and characterized for its physicokinetic properties. The purified enzyme appeared to be a monomeric structure with native molecular weight about 60 kD. The enzyme exhibited optimum pH 5.5 and pI 4.0, meso-thermostability and high temperature optimum (55°C) for catalytic activity, with activation energy of 6.8 kcal Mol^?1. The substrate saturation kinetics studies of the enzyme revealed a Michaelis–Menten constant (K_m) of 0.25 mM for pNPG and catalytic efficiency (K_cat/K_m) of 52,400 M ^?1 s^?1, respectively. Substrate specificity of the enzyme was restricted to β-linked gluco-, manno- and fuco-conjugates. The gluconolactone inhibition insensitivity was evident from its very low inhibition at millimolar inhibitor concentrations. Interestingly, the enzyme showed geraniol transglucosylating activity with pNPG as glucosyl donor but not with cellobiose. The catalytic activity of the enzyme has been reported to be novel with respect to its activity and preferences from a medicinal plant resource.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Properties of glutamate dehydrogenase from Beta vulgaris

Glutamate-NAD oxidoreductase, E.C. 1.4.1.3 (GDH), from seedlings of Beta vulgaris cv. Rota, Jahnsch Peragis Comp., was enzymatically characterized. This enzyme with molecular weight of 2.6 × 10⁵ has a pH optimum of around 8 for animation of α-KGA and around 9.5 for the desamination of glutamate. The apparent K_m for α-KGA is 6.7 × 10^?4M, for NH₃ 2.5 × 10^?3M, for NADH 3.2 × 10^?5M and for NAADPH 5.5 × 10^?4M. NAD¹ inhibits the reaction non-competitively when NADPH serves as substrate. The apparent K¹ is 4.5 × 10^?4M. The data are discussed on relation to the properties of GDH from other plant sources.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

Immobilization of β-glucosidase from Scytalidium lignicola on Chitosan

Chitosan was found to be a better support than alginate beads for immobilization of β-glucosidase from Scytalidium lignicola. The optimum concentration of glutaraldehyde for enzyme immobilization was 0.2%. Immobolized β-glucosidase was more able in the pH range of 3–6. Immobilized β-glucosidase retained about 70% of its activity at 50%C after 72 h of incubation while free enzyme lost most of its activity. The log of activity retained vs time was a straight line with free enzyme but was curved for immnobilized enzyme. Lineweaver-Burk plots of free and immoblized β-glucosidase gave K_m values of 2 × 10⁻⁴ M and 5.5 × 10⁻⁴ M for p-nitrophenyl β-d-glucopyranoside, respectively. Addition of immobilized β-glucosidase to a saccharification system gave a 30% increase in reducing sugar availability compared to free enzyme addition and was at least 4 times reusable without appreciable loss in enzyme activity.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.     

Isolation and some properties of two extracellular β-glucosidases from Trichosporon adeninovorans

The yeast Trichosporon adeninovorans secretes two multiple forms of β-glucosidase at a high rate if grown in a medium containing cellobiose. Following mutagenesis a mutant strain resistant to 2-deoxy-D-glucose was selected. This strain produced more β-glucosidase activity and had acquired a strong resistance against repression by glucose. The β-glucosidases were separated one from each other by chromatography on hydroxylapatite and by gel filtration. Both enzymes have similar properties. The optimal temperature for their activity was 60 to 63°C and the enzymes displayed highest activity at pH of 4.5. The molecular weight of β-glucosidase I was found to be 570,000 and that for β-glucosidase II was 525,000. The K_m value for cellobiose was determined to be 4.1 mM for β-glucosidase I and 3.0 mM for β-glucosidase II.     

Expression and characterization of a glucose-tolerant β-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and β-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a β-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most β-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K _m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10⁻², 5.6 × 10⁻², and 5.3 × 10⁻² mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and β-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.

     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

The Influence of pH on the Cadmium-Repressed Growth of the Alga Coelastrum proboscideum

The inhibition of growth by different concentrations of CdCl₂ in the range 4,5 × 10^?7 to 5.6 × 10^?7M was studied in the green alga Coelastrum proboscideum Bohlin in inorganic media at pH 4.3, 5.3 and 6.2. The factorial destgn of the experiments was evaluated as an analysis of 2² factors. Below pH 4.0 and above pH 6.5 growth was depressed without adding Cd. Cd concentrations exceeding 5.6 × 10^?8M reduced algal growth significantly with a 50% inhibition at 5.6 × 10^?7M Cd. The Cd concentration of 5.6 × 10^?7M was less toxic at pH 6.2 than at pH 5.3 and 4.3, thus revealing a negative interaction between protons and Cd.     

Stimulation of Growth of Peppermint (Mentha piperita) by Phosfon, a Growth Retardant

The shoot growth and fresh weight of Mentha piperita grown in soil were stimulated at concentrations of 1.26 × 10^?5M to 7.77 × 10^?4M phosfon (2,4-dichlorobenzyl tributyl phosphonium chloride) while higher concentrations resulted in retardation of growth. Concentrations of 6.30 × 10^?7M to 3.78 × 10^?5M caused retardation of growth in mineral nutrient solution, and even death at the highest concentrations. However, when the M. piperita plants were grown in mineral nutrient solutions at concentrations of phosfon which had been sequentially lowered from 2.52 × 10^?8M to 2.52 × 10^?12M, the shoot growth and fresh weight were stimulated as in the case of plants grown in phosfon treated soil.     

Calorimetric Glucose Determination by Glucose Oxidase —Methylene Blue

The reaction between glucose and methylene blue, catalyzed by glucose oxidase (GOD)was analysed calorimetrically. The amount of heat produced under saturating methylene blue concentrations ( > 10^?2 mol/1)was measured with glucose concentration and time as parameters (kinetic procedure) Kinetic constants (pseudo one substrate kinetics) were derived from the experimental data: K_M(glucose)= 1.18 × 10^?3 mol/l and V_max = 0.085 J/mg GOD min (3.89 · 10^?6 mol/mg GOD min) Comparison of caloric with optical measurements gave an enthalpy of reaction of 22.52 kJ/mol. Considering the observed substrate inhibition, glucose determinations are possible up to glucose concentrations of 0.1 mol/l.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.