Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: I. Effect of nucleotides and metal ions

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in microsomal fractions from chicken liver and hepatoma, induced by the leukosis virus strain Mc-29, using asialofetuin as the substrate acceptor of N-acetylneuraminic acid. The effect of some nucleotides and metal ions on the enzyme activity was investigated. Kinetic studies revealed that the Km values toward asialofetuin at a saturation concentrations of CMP-N-acetylneuraminic acid for both liver and hepatoma enzymes are very closed, while V value was lower for the tumor enzyme. The liver and hepatoma enzymes have no exogenous Mn cations requirement and are inhibited by CTP, CMP and ATP. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.24 mM for the liver and 0.16 mM for hepatoma enzyme, respectively.     

Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.     

Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.     

Studies on glycosyltransferases in fusion-defective conA-resistant L6 rat myoblast cell lines

1. Sialyl- and galactosyl-transferase activities were determined in wild type and conA-resistant L6 rat myoblasts with substrates derived from fetuin, alpha 1-acid glycoprotein and bovine submaxillary mucin; fetuin was the best acceptor for both enzyme activities, whereas the mucin did not act as an acceptor. 2. The optimum pH for sialyltransferase was 6.6 in both cell lines. 3. The optimum pH for galactosyltransferase in the wild type cell line was 6.2 which was slightly higher than the value of 5.8 found for the conA-resistant cells. 4. Values for Km for both enzyme activities increased five to ten-fold in the variant cell line with both acceptors. 5. The main sialyltransferase activity was the Gal beta 1----4GlcNAc alpha 2----3sialyltransferase for N-linked chains. The galactosyltransferase was most likely the enzyme that is responsible for the synthesis of the Gal beta 1----4GlcNAc structure.     

Characterization of the purified hyaluronan synthase from Streptococcus equisimilis

Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was approximately 40% more active in 25 mM, compared to 50 mM, PO(4) in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK(a) values at pH 6.6 and 11.8. As the pH was increased from approximately 6.5, both K(m) and V(max) increased until pH approximately 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Delta9) fatty acids. The apparent E(act) for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V(max) at 21, 6.5, and 3.5 mM, respectively. The apparent K(i) values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.     

Characterization of a CMPNeuAc: lactosylceramide alpha 2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells

1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.     

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.     

Species-specific aggregation factor in sponges. Sialyltransferase associated with aggregation factor.

The sialyltransferase (= glycoprotein-sialic acid transferase) was studied in the sponge Geodia cydonium, a mesozoan organism. The experiments were performed both in intact cellular and in isolated enzyme systems. It is shown, that desialylated cells show a lower aggregation potency than the controls. During aggregation enzymic sialylation of desialylated sponge cells occurs in the presence of an aggregation factor, which is associated with a high molecular weight particle. The sialylation process is temperature-dependent and can be inhibited by N-ethylmaleimide. Sialylation occurs predominantly at a distinct cell surface component, the aggregation receptor. The sialyltransferase was isolated and purified by the following steps: Sepharose 4B, CM-cellulose, Nonidet treatment, and Sephadex G-100. By this procedure the enzyme was purified 680-fold with a 31% yield. The sialyltransferase is originally associated with the high molecular weight particle also carrying the aggregation factor. In the last step the aggregation factor was separated from the sialyltransferase. The enzyme catalyzes the transfer of sialic acid from CMP-sialic acid to the desialylated aggregation receptor. The molecular weight of the sialyltransferase has been determined to be 52,000. Kinetic studies revealed no lag phase and a dependence on enzyme concentration. The purified transferase has a pH optimum of 7.75 and requires 200 mM NaCl for activity. No requirement for Mg2+ or Ca2+ could be observed. The reaction is inhibited by 10 micronM N-ethylmaleimide.     

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells

Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.     

Control of Uridine Diphosphate-Glucose Dehydrogenase Synthesis and Uridine Diphosphate-Glucuronic Acid Accumulation by a Regulator Gene Mutation in Escherichia coli K-12

Uridine diphosphate (UDP)-glucose dehydrogenase, the enzyme that converts UDP-glucose to UDP-glucuronic acid, was derepressed in a mucoid (capR9) strain of Escherichia coli K-12 and repressed in a nonmucoid (capR(+)) strain. A nonmucoid mutant (strain MC 152; capR9 non-2) derived from the mucoid strain accumulated large quantities of nucleotides. Among these nucleotides, UDP-glucuronic acid was identified as well as guanosine triphosphate and an adenosine diphosphate-sugar. UDP-glucose dehydrogenase was still derepressed in strain MC 152. When the nonmucoid mutant was transduced to the wild-type state for this regulator gene (capR(+)), the transductant was found to accumulate less total nucleotides, and the accumulation of UDP-glucuronic acid was abolished. UDP-glucose dehydrogenase was repressed in the capR(+)non-2 strain but not to the same extent that it was in the capR(+) strain.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms

A rat brain Golgi sialyltransferase activity capable of the differentiation-dependent control of N-CAM sialylation state is described. The specific activity of Golgi sialyltransferase was found to be developmentally regulated with respect to both endogenous and exogenous protein acceptors, with a particular elevation on postnatal days 10-12 when the heavily sialylated or 'embryonic' form of N-CAM is re-expressed. The subsequent developmental decrease in activity was associated with a significant decrease in apparent Km for the CMP-NeuNAc substrate, but not for the asialofetuin exogenous acceptor, which could not be attributed to the temporal expression of an endogenous competitive inhibitor. The apparent Vmax remained constant for CMP-NeuNAc but was significantly reduced for asialofetuin. Sialyltransferase activity, which was optimal at pH 7.0-7.5, was also modulated by various cations. Zinc abolished enzyme function, in contrast to ferric ions which stimulated activity fourfold-sevenfold. The marked activation of the adult form of the enzyme by potassium and magnesium ions, together with the alterations in kinetic constants, suggested this activity to be distinct from that derived from postnatal day-12 tissue. The kinetics of [14C]sialic acid incorporation into immuno-precipitated N-CAM demonstrated the individual polypeptides to be sialylated, possibly by addition of polysialosyl units, in a developmental sequence. The presence of four distinct sialyltransferase activities was demonstrated by non-denaturing gel electrophoresis followed by solid-phase enzyme assay. These isoforms were temporally expressed during development, two being correlated with the postnatal reexpression of the 'embryonic' form of N-CAM.     

Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).     

Glycosylation of kappa-casein. I. Localization and characterization of sialyltransferase in bovine mammary gland.

A sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) which attaches N-acetylneuraminic acid to the terminal end of the carbohydrate chain of kappa-casein was found to be concentrated in Golgi apparatus-enriched fractions of bovine mammary gland. Maximum sialyltransferase activity was obtained at pH 5.5 and 37 degrees C in the presence of 1 mM dithiothreitol and Triton X-100. A Km of 0.19 mg asialo-kappa-casein/ml (0.01 mM) was obtained for the sialyltransferase. Native kappa-casein also served as acceptor for N-acetylneuraminic acid transferase of Golgi apparatus-enriched fractions although at a slower rate than did asialo-kappa-casein. The sialyltransferase has a divalent cation requirement for maximum activity which was best satisfied by the presence of 10 mM Mn2+.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Effect of divalent cations on the inhibition of alkylsulfatase induction and the generation of ATP in Pseudomonas aeruginosa after exposure to exogenous uridine 5'-triphosphate.

In the absence of added Mg2+, alkylsulfatase induction in resting cells of Pseudomonas aeruginosa was inhibited 17% by exogenous 0.05 mM UTP. Under these conditions, the cells converted UTP to ATP and rapid degradation of these nucleotides did not occur. In the presence of 0.73 mM Mg2+, 0.05 mM UTP repressed the synthesis of the enzyme by 71%. Under these conditions, the cells rapidly degraded both ATP derived from UTP as well as residual UTP. In the presence of Mg2+ and 0.1 mM UTP, full repression of alkylsulfatase formation occurred whereas Mg2+-depleted cell suspensions were still capable of synthesizing 47% of the enzyme under these conditions compared with control levels. The inhibition of alkylsulfatase induction was highly specific for UTP. Some inhibition was observed with exogenous uracil, uridine, and pyrophosophate but only at concentrations greater than 1.0 mM. Exogenous UMP and UDP (2mM) had no effect.     

Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.

A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.