Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells
Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells
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DOI：
10.1016/s1357-2725(00)00036-4
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发表时间：
2000-09-01
影响因子：
4
通讯作者：
Lagrou, AR
中科院分区：
文献类型：
--
作者：
De Busser, HM;Van Dessel, GAF;Lagrou, AR
以N-乙酰-S-法呢基-L-半胱氨酸和N-乙酰-S-香叶基-L-半胱氨酸为人工底物，以粗细胞匀浆为酶源，检测了牛肾上腺髓质嗜铬细胞中特异性异戊烯基半胱氨酸羧甲基转移酶的存在。从Michaelis-Menten动力学计算出下列常数：对于N-乙酰基-S-法尼基-L-半胱氨酸，k（m）为90 μ M，V-max为3 pmol/min/mg蛋白质;对于N-乙酰基-S-香叶基香叶基-L-半胱氨酸，K-m为52 μ M，V-max为3 pmol/min/mg蛋白质。两种底物在pH 7.4-8.0范围内均被甲基化至最佳程度。甲基化活性线性增加至20分钟孵育时间，并且在至少160 μ g蛋白质时呈剂量依赖性。Sinefungin和S-腺苷高半胱氨酸都引起了显着的抑制，也在较小程度上没有法尼基硫代乙酸，脱氧甲硫基腺苷和3-deaza-adenosine。效应子研究表明，甲基转移酶活性的变化取决于浓度和化学性质的阳离子存在。一价阳离子略有刺激，而二价金属离子表现出不同的抑制作用。螯合剂EDTA和EGTA的刺激作用验证了阳离子的抑制作用。巯基试剂抑制甲基化，但程度不同：Hg 2+离子：100%，N-乙基马来酰亚胺：30%，二硫苏糖醇：0%和单碘乙酸：20%。由于底物的疏水性，温育混合物中必须包括二甲基亚砜（< 4%;在更高的浓度下仍有中度抑制）。测试的洗涤剂在不同程度上影响甲基转移酶活性。证实了甲基转移酶的膜结合特性。(C)2000爱思唯尔科技有限公司版权所有。
Chromaffin cells fi om bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: k(m) 90 muM and V-max 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K-m 52 muM and V-max 3 pmol/min pel mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH ranee 7.4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 mug of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg2+-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture ( < 4%; still moderate inhibition at more elevated concentrations), The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed. (C) 2000 Elsevier Science Ltd. All rights reserved.