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).     

Interaction between gamma-carboxyglutamic acid and glutamate dehydrogenase

The amino acid γ-carboxyglutamic acid, recently discovered in some vitamin K-dependent blood-clotting factors, shows interesting kinetic effects on glutamate dehydrogenase. It is not metabolized by the enzyme; it is a powerful competitive inhibitor (K_i = 3.8 × 10^?4 m) with respect to NAD⁺ and glutamate. On the other hand the reverse reaction is activated by γ-carboxyglutamate, both K_m and V being altered; this effect is additive with the well-known activating effect of ADP.     

Recent advances in the in vivo and in vitro metabolism of retinoic acid.

Retinoic acid, a natural metabolite of retinol, has previously been shown to be capable of supporting growth and maintaining proper differentiation in epithelial tissues. Recently, investigation into the in vivo and in vitro metabolism of retinoic acid in hamsters, using both tracheal organ culture and subcellular preparations derived from intestinal mucosa, liver, and testis, has revealed the production of several metabolites more polar than the parent compound. Two of the early products of this metabolic pathway have been identified as 4-hydroxy- and 4-keto-retinoic acid. The formation of these metabolites is maximal in vitamin A-deficient hamsters that have been pretreated with retinoic acid and in vitamin A-normal animals. This fact, together with the decreased biological activity of the two compounds relative to retinoic acid in a tracheal organ culture assay, suggested that oxidative attack at carbon-four of the cyclohexenyl ring may be the first step in the elimination of retinoic acid from tissues. In addition, observations both in vivo and in vitro indicate that all-trans- and 13-cis-retinoic acid at low concentrations may be sharing a common metabolic pathway that includes an isomer of 4-keto-retinoic acid.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

Analysis of calorimetric data for isodesmic self-associating systems.

ATP and respiration (NADH)-driven NAD(P)⁺ transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P)⁺ transhydrogenase (reduction of acetylpyridine NAD⁺ by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P)⁺ transhydrogenase. It was found that the K_m values for acetylpyridine NAD⁺ and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μm) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μm), respectively, but the maximum velocity at infinite acetyl pyridine NAD⁺ and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P)⁺ transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD⁺ levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P)⁺ transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD⁺ and NADPH sites.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Genetic Recombination — Understanding the Mechanism : by H.L.K. Whitehouse Wiley; Chichester,New York, 1982

Interaction of peanut agglutinin with MeUmbβGalβ(1→3)GalNAc was followed with the stopped-flow technique. The mechanism is a simple bimolecular association with k₊ = 7.1 × 10³ M^?1. s^?1 and k_? = 0.24 s^?1 at 25°C. The very slow dissociation rate of the complex strongly supports earlier conclusions that the combining site of peanut agglutinin is complementary to the Galβ(1→3)GalNac structure.     

Mannitol Biosynthesis in Pyrenochaeta terrestris I. D-Maunitol-1-Phosphate: NAD Oxidoreductase

Cell-free extracts of mycelial mats of Pgrenochaeta terrestris grown in stationary culture on synthetic glucose or sucrose - salts liquid media contained D-mannitol-1-Phosphate:NAD oxidoreductase (EC 1.1.1.17) activity. Greatest activity occurred early in the growth period. The optimum pH for the reduction of NAD⁺ in the presence of Fru-6-P was 7.4–7.5 while the optimum pH for the oxidation of NADH in the presence of Mtl-1-P was 8.1–8.2. The enzyme was stabilized to some extent in Tris-maleate buffer, pH 7.5, and by the addition of 10% (NH₄)₂SO₄, to this buffer. A 10- to 16-fold purification was attained by a combination of (NH₄)₂SO₄ fractionation and gel filtration on Sephadex G-100. The enzyme was relatively specific in its substrate and coenzyme requirements. The Km values were determined as: Fru-6-P - 3 × 10^?4 M, Mtl-1-P - 1 × 10^?4 M, and NAD⁺ and NADH - 3 × 10^?5 M.     

Metabolisme du γ-aminobutyrate chez Agaricus bisporus III. La succinate-semialdehyde:NAD(P)+ oxydoreductase

Metabolism of γ-Aminobutyrate in Agaricus bisporus. III. The Succinate-Semialdehyde: NAD (P)⁺ Oxidoreductase. The succinate-semialdehyde:NAD(P)⁺ oxidoreductase (E.C. 1.2.1.16) is responsible for the second step in the catabolism of γ-aminobutyrate: the irreversible enzymatic conversion of succinic semialdehyde (SSA) to succinate. Succinate semialdehyde dehydrogenase was extracted from mitochondrial fraction of fruit-bodies of Agaricus bisporus Lge. The mitochondrial pellet was sonicated and centrifuged at 110,000 g; the supernatant obtained was designated the “crude extract”. The enzyme was extremely unstable on storage, unless 1 mM EDTA and 20% glycerol were added. Kinetic studies were carried out at 30°C, and the formation of NADH or NADPH was followed by measuring increase of absorbance at 340 nm with a spectrophotometer. The dehydrogenase was completely inactive when the reaction was run in the absence of thiol and was more active with NAD⁺ than with NADP⁺. In the “crude extract” the activity with NADP⁺ had a pH optimum between 8.6 and 9.1 and the K_m values for SSA and NADP⁺ were 2.0 × 10^?4M and 1.4 × 10^?4M respectively. The pH optimum with NAD⁺ was found between 8.6 and 8.8 and the K_m value for SSA is 4.8 × 10^?4M and for NAD⁺ 2.0 × 10^?3M. With NAD⁺, the kinetic values (pH, K_m) of the “crude extract” chromatographed on hydroxylapatite were unchanged. Inhibition by thiamine pyrophosphate (TPP) was uncompetitive with respect to NAD⁺, those by malate, ATP, ADP and NADPH non-competitive and that by NADH competitive. These results and the fact that activity with NAD⁺ was lost more slowly than with NADP⁺ indicate the possibility of at least two mitochondrial succinate-semialdehyde dehydrogenases, even though the activities of this enzyme assayed with NAD⁺ and NADP⁺ respectively were not able to be separated from each other by hydroxylapatite column chromatography. Some speculations on the metabolic regulation of this dehydrogenase and considerations on the significance of these results in the physiology of respiration in Agaricus bisporus Lge are given.     

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10^?8 to 3 × 10^?6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10^?7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.     

The effect of cadmium ions and cadmium-metallothionein on the activities of phospholipid-synthesizing enzymes of rat liver microsomes in vitro

The effects of cadmium ions or cadmium-metallothionein on the activities of acyl-CoA:1acyl-sn-glycerol 3-phosphoric acid or 1-acyl-sn-glycero 3-phosphocholine acyltransferase of rat liver microsomes have been studied, in vitro. Cadmium ions were found to cause a noncompetitive type inhibition of these two acyltransferases. The K_i values were calculated, and found to be smallest (1.7 × 10^?5m) for palmitoyl-CoA and greatest (1.0 × 10^?4m) for linoleoyl-CoA, among the several fatty acyl-CoA's tested on the 1-acyl-sn-glycerol 3-phosphoric acid acyltransferases. With the 1-acyl-sn-glycero 3-phosphocholine acyltransferase, the K_i values were found to be smallest for the plamitoyl-CoA acyltransferase (3.8 × 10^?5m) and largest for thearachidonoyl-CoA acyltransferase (1.1 × 10^?4m). In contrast, mouse liver cadmium-metallothionein, including 4 mol of cadmium and 2 mol of zinc in one molecule of metallothionein, was not found to be inhibitory or rather stimulative on the above two acyltransferases at the same concentration of cadmium tested in the cadmium ion inhibitor experiments. The above results demonstrate that there is a strong and irreversible inhibition by cadmium ions on acyl-CoA acyltransferases, but that when cadmium acts on the enzyme in the form of a cadmium-metallothionein complex, the inhibition effect does not occur. These findings may reflect differing degrees of toxicity of these two types of cadmium compounds in mammalian tissues.     

Preparation,characterization, biological activity and metabolism of all-trans retinoyl fluoride

All-trans retinoyl fluoride was prepared by treating all-trans retinoic acid with diethylaminosulfurtrifluoride. The crystalline product, which was characterized by melting point, infrared, ¹H-NMR, ¹⁹F-NMR and elementary analysis, showed λ_max at 382 nm in hexane (ε = 4.98·10⁴ M^?1·cm^?1) and at 392 nm in methanol (ε = 4.60·10⁴ M^?1·cm^?1). Its biological activity in the rat growth assay, relative to all-trans retinyl acetate, was 22% ± 10%. Upon oral administration for 5 days to vitamin A-depleted rats, retinoyl fluoride (1020 μg) was rapidly metabolized to a polar metabolite fraction and, in the intestine, to an unstable retinol-like metabolite, purpotedly 15-fluororetinol. Upon administering intraperitoneally smaller doses (47–94 μg) of [11-³H]retinoyl fluoride, which was synthesized from [11-³H] retinoic acid, radioactive retinoic acid was noted in the liver and plasma but not in the intestine. As expected, a radioactive polar fraction appeared in the intestine and liver, but radioactive retinol, retinyl ester and some common oxidation products were not detected. Of the administered radioactivity, 72% was excreted in the urine, and only 4% was found in the feces over a 7-day period. Hydrolysis of the urine gave a radioactive fraction with a polarity similar to that of retinoic acid. Retinoyl fluoride also reacts readily with glycine to yield N-retinoyl glycine. Thus, the biological activity of retinoyl fluoride can be attributed to the formation of retinoic acid, probably by way of N-retinoyl derivatives. A possible pathway for its metabolism is presented.     

The biological activity of rat intestinal retinoic acid metabolites in the vaginal smear assay

Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-³H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [³H]retinoic acid. The biological activity of all discernible metabolities was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-β-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action.     

Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD⁺ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD⁺/UDP-GlcA and MoeE5/NAD⁺/UDP-glucose, determined at 1.48 Å and 1.66 Å resolution. The cofactor NAD⁺ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD⁺, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.     

Plant pyruvate dehydrogenase complex: analysis of the kinetic properties and metabolite regulation

The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent K_m values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD⁺, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD⁺, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD⁺. Among various metabolites tested, only hydroxypyruvate (K_i = 0.11 mM) and glyoxylate (K_i = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg²⁺? or Ca²⁺-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.     

Stearyl-CoA desaturase of bovine mammary microsomes

Stearyl-CoA desaturase from the microsomal fraction of lactating bovine mammary tissue had a specific activity of 0.4 nmoles oleate formed min^?1 mg^?1 protein. NADH was required for desaturase activity. However, oxidized NAD⁺ and NADP⁺ supported measurable desaturase activity. K_m values for stearyl-CoA and NADH were 25.0 μm and 3.0 μm, respectively. Desaturase was depressed by increasing concentrations of other acyl-CoA esters, i.e., palmityl-CoA and oleyl-CoA (>10 μm). Sn-1,2 diglycerides (1–2.0 μm) depressed desaturase slightly in the order 0–20%, as did l-α-glycerolphosphate (0.2–3.6 μm). 1-Acyl-sn-glycerol-3-phosphorylcholine (>0.1 μm) depressed desaturase activity markedly. Sonication of the microsomal preparation stimulated desaturase activity. The addition of ethanol depressed desaturation, and EDTA inhibited desaturation. Palmityl CoA was equally desaturated by the microsomes. The acyl-CoA desaturase was very stable when stored at ?30 °C as a freeze-dried microsomal preparation, i.e., activity was retained after 12-month storage.Labeled stearate and oleate were isolated as esters (triglycerides and phospholipids) and as free fatty acids, indicating the presence of acyl transferases and acyl-CoA hydrolase in mammary microsomes.     

Determination of the viscometric constants for chitosan

The viscometric constants a and K_m in the Mark-Houwink equation have been determined for chitosan in 0.1 m acetic acid 0.2 m sodium chloride solution, using the approach of Sharples and Major. The number-average molecular weights were determined by absorbance measurements on solutions of the phenylosazone derivatives. The values obtained a = 0.93, K_m = 1.81 × 10^?3 cm³ g¹ differ considerably from those reported previously by Lee but are in agreement with values found for other ionic polysaccharides having related β-(1 → 4)-linked structures.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.