Control of rabbit liver fructose-1, 6-diphosphatase activity by magnesium ions.

EDTA at a concentration of 1 muM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3.1.3.11] in the presence of 5 mM Mg2+ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg2+. A double-reciprocal plot of the data gave the Km values corresponding to the two linear regions. They were 0.19 and 0.83 mM at pH 7.5, and 0.055 and 0.83 mM at pH 9.1. In the presence of 5muM EDTA a sigmoidal curve was obtained for Mg2+ activation in the range of noninhibitory Mg2+ concentrations at pH 7.2. The apparent Km value for Mg2+ was 0.15 mM, and the Hill coefficient was 2.0. At pH 9.1 cooperativity among the Mg2+ sites disappeared, and the apparent Km value for Mg2+ was 0.055 mM. These Km values at pH 7.2 or 9.1 corresponded to the smaller of the biphasic Km values obtained without EDTA. In the absence of EDTA, no inhibition by Mg2+ was observed in the Mg2+ concentration range below 10 mM. In the presence of EDTA, the enzyme was inhibited markedly by Mg2+ at concentrations above 0.5 mM at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the Km value for Mg2+ activation and on the Mg2+ inhibition contributed to an apparent shift of the pH optimum for activity induced by EDTA. Cooperative interaction among fructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximatley 1.8, and the apparent Km value for the substrate was 0.74 muM. EDTA appears to make liver fructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg2+ serves as a regulator for the enzyme activity.     

Fructose 1:6 bisphosphatase activity in the liver and testis of rats and goats

1. The activity of the enzyme Fructose 1:6 Bisphosphatase (FDPase) was studied in the liver and testis of adult goats and rats. No significant difference in the enzyme activity was observed between liver and testis of rats. Highly significant differences (P less than 0.01) were observed between the activity of goat liver and goat testis, goat liver and rat liver, goat testis and rat testis; the activities being higher in goat tissues. 2. Homogenization of the tissues with water, 0.05 M lactate buffer (pH 3.5), 150 mM KCl and 0.34 M sucrose yielded highest activity with water and lactate buffer followed by Sucrose and KCl. 3. 10 microM of Fe2+ and 45 microM of Zn2+ decreased the enzyme activity of rat testis by 39% and 93% respectively. 4. The rate of hydrolysis of FDP with respect to time in rat liver and testis was a first order reaction. Linear kinetics of the substrate hydrolysis was observed up to 90 min. No substrate inhibition of the enzyme activity was observed up to 50 microM of the substrate. Km of rat liver and testis FDPase were 8.3 microM and 10.5 microM respectively.     

Appearance of sedoheptulose 1,7-diphosphatase activity on conversion of chloroplast fructose 1,6-diphosphatase from dimer form to monomer form.

Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg⁺⁺) but retained ferredoxin-dependent activity (measured in the presence of Mg⁺⁺ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor.     

蛇肌果糖1，6－二磷酸酯酶果糖2，6－二磷酸和底物抑制的结合部位

在果糖１,６—二磷酸酯酶中果糖２,６—二磷酸可能与底物抑制的作用方式不同,因为蛇肌果糖１,６－二磷酸酯酶ｐＨ９．２的活性受到果糖２,６－二磷酸的抑制,而不受高浓度底物的影响。Ｋ＋能增强果糖２,６—二磷酸对酶活性抑制,并能较大程度地解除过量底物的抑制。快反应流基修饰酶不再受较低浓度果糖２,６—二磷酸的抑制,但高浓度果糖２,６—二磷酸仍能抑制酶活性,其ＩＣ５０增大４０倍。修饰酶受底物抑制的阈值不变。为胰蛋白酶或枯草杆菌蛋白酶限制性酶解的果糖１,６—二磷酸酯酶受过量底物和果糖２,６—二磷酸抑制的行为也不相同。以上结果可能提示在蛇肌果糖１,６—二磷酸酯酸中存在既有别于ＡＭＰ,又有别于过量底物的结合部位。     

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5–8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2 h, followed by adjustment to a pH in the range of 6.5–8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.     

Purification of the native form of tyrosine aminotransferase from rat liver

A procedure that provides a homogeneous, native form of tyrosine aminotransferase (l-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver in exceptionally high yield is described. This goal is accomplished by rapidly inactivating the lysosomal converting factor that generates two additional, lower-molecular-weight forms of tyrosine aminotransferase, and by separating the native enzyme from the altered forms by chromatography on carboxymethyl-Sephadex C-50 and an optional hydroxylapatite step. Homogeneity appears to be achieved after the carboxymethyl-Sephadex C-50 step, and the final yield of the enzyme exceeds 30%.