Rao K N
Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Bombay, India.
Indian J Biochem Biophys. 1995 Feb;32(1):11-20.
The effects of varying concentrations of urea, thiourea and guanidine hydrochloride on the enzyme activity and the isoenzymic polypeptide association of pteroylpoly-gamma-glutamyl hydrolase (EC 3.4.22.12) from chicken liver were studied. Incubation of the enzyme at 4 degrees C with low concentrations of the buffered (100 mM sodium acetate containing 1% ascorbate, pH 4.1) solutions of urea (0.55 M) and guanidine hydrochloride (0.05 M) resulted in stimulation (5- and 2-fold respectively) of the activity of the enzyme whereas at higher concentrations of the denaturants (6 M urea, 1 M thiourea or 2 M guanidine hydrochloride) the enzyme was completely inactivated. However, there was no enzyme activation in response to thiorea treatment. Under specific denaturing conditions the association of two isoenzymic polypeptides was studied. The 0.55 M urea- and 0.05 M guanidine hydrochloride-activated enzyme displayed its disaggregated nonidentical polypeptides I and II (M(r) = 41,000 and 17,300 respectively) on Sephadex G-100 gel filtration, SDS-PAGE and sedimentation analyses. The 8 M urea- and 3 M guanidine hydrochloride-inactivated enzyme on the other hand exhibited a single protein aggregate species of an M(r), 57,000 like the native enzyme. Both unmodified native enzyme and the pCMB-modified PtepolyGlu hydrolase responded similarly to these denaturants. The two constituent active polypeptides polyp-I and polyp-II of the heterodimeric gamma-glutamyl glutamyl hydrolase are dissociated in the presence of 0.55 M urea as evident from the PAGE analyses. Some catalytic properties of the activated enzyme were studied and compared with those of the native enzyme. The urea-activated enzyme displayed a shift in the second pH optimum of the double pH-activity profile (optima at pH 4.1 and pH 5.2) from pH 5.2 to pH 6.0. The activated enzyme has a Km value of 0.59 x 10(-6) M (Vmax, 0.10) for 5-CH3-H4PteGlu4 while the native enzyme has the Km of 0.83 x 10(-6) M (Vmax, 0.03) for this substrate. When the reaction mixtures were incubated with the urea-activated gamma-glutamyl hydrolase, a maximum stimulatory effect on the enzyme activity was observed with the bivalent metal ion Ca2+ whereas the most potent inhibitory effect was observed with the trivalent anion citrate.
研究了不同浓度的尿素、硫脲和盐酸胍对鸡肝蝶酰多聚 -γ- 谷氨酸水解酶（EC 3.4.22.12）的酶活性和同工酶多肽缔合的影响。将该酶于4℃下与低浓度的缓冲溶液（含1%抗坏血酸的100 mM醋酸钠，pH 4.1）中的尿素（0.55 M）和盐酸胍（0.05 M）一起温育，结果酶活性分别受到刺激（分别为5倍和2倍），而在较高浓度的变性剂（6 M尿素、1 M硫脲或2 M盐酸胍）作用下，酶完全失活。然而，硫脲处理未引起酶的激活。在特定的变性条件下研究了两种同工酶多肽的缔合。经0.55 M尿素和0.05 M盐酸胍激活的酶在Sephadex G - 100凝胶过滤、SDS - PAGE和沉降分析中显示出其解聚的不同多肽I和II（相对分子质量分别为41,000和17,300）。另一方面，经8 M尿素和3 M盐酸胍失活的酶表现出与天然酶类似的单一相对分子质量为57,000的蛋白质聚集体。未修饰的天然酶和经pCMB修饰的蝶酰多聚谷氨酸水解酶对这些变性剂的反应相似。从PAGE分析明显看出，在0.55 M尿素存在下，异二聚体γ- 谷氨酰谷氨酸水解酶的两个组成活性多肽polyp - I和polyp - II发生了解离。研究了激活后酶的一些催化特性并与天然酶的进行了比较。尿素激活的酶在双pH - 活性曲线（最适pH为4.1和pH 5.2）的第二个最适pH处从pH 5.2移至pH 6.0。激活后的酶对5 - CH3 - H4PteGlu4的Km值为0.59×10⁻⁶ M（Vmax为0.10），而天然酶对该底物的Km值为0.83×10⁻⁶ M（Vmax为0.03）。当反应混合物与尿素激活的γ- 谷氨酰水解酶一起温育时，二价金属离子Ca²⁺对酶活性观察到最大刺激作用，而三价阴离子柠檬酸盐观察到最有效的抑制作用。
