Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

6-Phosphogluconate dehydrogenase in cell free extracts of Escherichia coli K-12

Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10^-2 m MgCl₂ produced maximal activity, whereas higher concentrations caused inhibition. The K _m values were 2.5×10^-4 m for 6-phosphogluconate and 2.5×10^-5 m for NADP⁺ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl₂ buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Characterization and delignification activity of a thermostable α-l-arabinofuranosidase from Bacillus stearothermophilus

Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and -l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl--l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (K _m) of 2.2 × 10^–4 m, maximum reaction velocity (V_max) of 11o mol min^–1 mg^–1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml^–1 of -l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin. Correspondence to: Eugene Rosenberg     

Red cell hydrolases

Summary A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing³H-acetylated hemoglobin (162 cpm/pmole) as a substrate. Two proteolytic enzymes having optimum activity at pH 3.4 and pH 7.4 were isolated from Sephadex G-100. The protease active at pH 3.4 was 75 times as active as the pH 7.4 enzyme and it was purified 182-fold over the original homogenate and characterized. A linear relationship for activity versus time and activity versus concentration of enzyme was found. The optimum temperature was 37°C and theK _m was 1×10^–5 m hemoglobin. No enzyme activation was observed with any cation studied and EDTA had no inhibitory effect; (10mm Fe⁺³ and Hg⁺² were inhibitory). The pH 3.4 protease was stable indefinitely at –20°C in 0.1% Triton X-100. Gel electrophoresis was performed on a sodium dodecylsulfate-mercaptoethanol enzyme preparation and two protein bands (mol. wt. 33,000 and 54,000) were evident for the Sephadex G-200 eluate containing the pH 3.4 protease.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). II. Erythrocyte glucose-6-phosphate dehydrogenase in arctic and silver foxes: Purification and properties

The functional properties of purified glucose-6-phosphate dehydrogenase (G6PD) from the erythrocytes of Arctic foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) were investigated. It was found that pH optima for G6PD range from 8.15 to 8.25 in Arctic foxes and from 10.2 to 10.4 in silver foxes. For G6P, the estimated K _m values were 74×10^–6 m (at pH 8.2) and 166×10^–6 m (at pH 10.2) in Arctic foxes and 58×10^–6 m (at pH 10.2) and 40×10^–6 m (at pH 8.2) in silver foxes. The K _m values for NADP were estimated as 62×10^–6 m (at pH 8.2) and 86×10^–6 m (at pH 10.2) in the Arctic foxes and 15×10^–6 m (at pH 10.2) and 12×10^–6 m (at pH 8.2) in the silver foxes. It was found that Mg²⁺ ions exert a significant activating effect on G6PD in the Arctic fox and do not affect appreciably its activity in the silver fox. The experimental data indicate that slight differences in the electrophoretic mobility of G6PD are associated with considerable functional differences in this enzyme between the two fox species.     

Zinc inhibition of chloride efflux from skeletal muscle ofRana pipiens and its modification by external pH and chloride activity

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles fromRana pipiens, the pH-dependent Cl^– efflux has an apparent pK _a near 6.4.The reduction of Cl^– efflux by external Zn²⁺ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl^– efflux was also examined.At pH 6.5 and a membrane potential of –22 mV, increasing external Cl^– activity from 0.108 to 0.28m decreased inhibition of the pH-dependent Cl^– efflux at all activities of Zn²⁺. The Zn²⁺ activity needed to reduce Cl^– efflux by half increased from 0.39×10^–3 to 2.09×10^–3 m. By contrast, external Cl^– activity had no measurable effect on the apparent pK _a of the pH-dependent efflux.At constant Cl^– activity less than 0.21m, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn²⁺ activities with either a slight increase or no change in the Zn²⁺ activity producing half-inhibition. In other words, for relatively low Cl^– activities, protection against inhibition of Cl^– efflux by low Zn²⁺ activities was obtained by raising, not lowering, external pH; this is not what is expected if H⁺ and Zn²⁺ ions compete at the same site to produce inhibition of Cl^– efflux. We conclude that Zn²⁺ and low pH inhibit Cl^– efflux by separate and distinct mechanisms.By contrast, the protection against Zn²⁺ inhibition produced by high external Cl^– activity (0.28m) was partially reversed by raising external pH from 6.5 to 7.5 at all Zn²⁺ activities. The half-inhibition Zn²⁺ activity decreased from 2.09×10^–3 to 0.68×10^–3 m.The results can be simulated quantitatively by a model in which single Cl^– channel elements are in equilibrium with sextets of associated single-channel elements, each sextet having a conductance six times that of a single-channel element. The association into sextets is promoted by OH^– or Cl^– binding to a control site on the single-channel elements. Both the single Cl^– channel element and the sextet of Cl^– channel elements are closed when this same control site instead binds ZnOH⁺. The sextet has a much higher affinity for ZnOH⁺ than does the single Cl^– channel element.     

Enzyme kinetics in cold water: characteristics of N-acetyl-β-d-glucosaminidase activity in the Antarctic krill,Euphausia superba,compared with other crustacean species

N-acetyl--d-glucosaminidase, a chitin-degrading enzyme, is highly active in the integument and digestive tract of euphausiids. The enzyme was used as a model to compare temperature-dependent enzymatic parameters of Antarctic krill, Euphausia superba, with those of a euphausiid species (Meganyctiphanes norvegica) found in both the Scandinavian Kattegat and the Mediterranean. Other species examined were an Antarctic isopod, Serolis polita, and a tropical crab, Ocypode ryderi. Enzyme isoforms of NAGase were isolated chromatographically. Temperature optimum (between 30 and 53 °C) and activation-energy (47–59 kJ·mol^-1) of isoenzymes were generally unrelated to genotypic cold adaptation. Although pH profiles were temperature-dependent, there was no apparent temperature-related control of activities by pH in the experienced physiological range. In contrast, apparent Michaelis constants showed minima at ambient water temperatures (total range: 0.1–0.6 mol·l^-1). Potentially, enzyme variants play a role in acclimatisation regulated by Michaelis constants. Apparently, the rate-limiting effects of polar temperatures are partly compensated in the Antarctic crustaceans by construction of enzymes with substrate affinities similar to those of species from warmer climates. The significance of apparent Michaelis constants in evaluating mechanisms of metabolic cold compensation is discussed. Necessary additional experimental approaches are highlighted.Abbreviations CPB citrate-phosphate buffer - FPLC fast protein liquid chromatography - K _m ^app apparent Michaelis constant - LC liquid chromatography - MW molecular weight - NAGase N-acetyl--d-glucosaminidase     

Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity

We purified a novel -glucosidase to homogeneity from an Escherichia coli recombinant transformed with the -glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known -glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O--d-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k _O) and the Michaelis constant (K _m) values at 55°C and pH 6.0 for sucrose were 54.6 s^–1 and 5.3 mm, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75°C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 m sucrose at 60°C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65°C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

Amino acid racemase with broad substrate specificity,its properties and use in phenylalanine racemization

Summary Broad substrate specificity amino acid racemase (EC 5.1.1.10) was purified from a crude extract of Pseudomonas putida SCRC-744 to near homogeneity. The enzyme has an isoelectric point of 7.6 and a molecular weight of 62,000–65,000. The enzyme showed a broad substrate specificity toward amino acids, utilizing d-glutamine as the best substrate. d-Phenylalanine acted as a substrate to 1% the velocity for d-glutamine. Maximal reaction velocities were observed at 50°–60°C and around pH 8. The apparent K_m values for d-glutamine and d-phenylalanine were 7.8 mM and 25.7 mM, respectively. Both enantiomers of phenylalanine were efficiently racemized by acetone-dried cells of P. putida SCRC-744.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Interactions of lanthanum with purified and intact cell acetylcholinesterase of electrophorus electricus

Summary The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl₃ produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl₃, initial activity was markedly increased. LaCl₃ exerted a marked, concentration-dependent inhibition of intact cell AChE.La³⁺ and Ca²⁺ appear to interact competitively. In the presence of both 10mm CaCl₂ and 0.1mm LaCl₃, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10^–4 m. Inhibition of intact cell enzyme by 0.1mm LaCl₃ was relieved by increasing the CaCl₂ concentration to 10mm at ACh concentrations less than 2 × 10^–4 m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.     

Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.     

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Summary A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ^–1 and with 6.6×10^–12 moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ^–1 and 1.0×10^–12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Chemical modifications of histidine residues in cytoplasmic asparate aminotransferase from beef kidney

Summary Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 × 10^–6 m tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 × 10^–5 m) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation.Diethylpyrocarbonate modifies three histidine residues per enzyme protomer and reduces the activity only 10%. These results suggest that the two histidine residues photoxidized through the sensitizer, are located in the active site of the enzyme, at least one of these appears to be involved in ketosubstrate binding. The other three histidines modified by diethylpyrocarbonate are likely located on the enzyme surface and are not involved in the catalytic activity of the enzyme.This work is part of a program supported by a grant from the Consiglio Nazionale delle Ricerche.