Kulbe K D, Bojanovski M, Lamprecht W
Eur J Biochem. 1975 Mar 17;52(2):239-54. doi: 10.1111/j.1432-1033.1975.tb03992.x.
Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.
来自牛肝的均一磷酸甘油酸激酶在278 nm处有最大紫外吸收（A 1%,1Cm 280等于6.7；Amax/Amin等于2.26；e280等于31.5 mM(-1)×cm(-1)）。该酶由约420个氨基酸残基组成，是一种微酸性蛋白质，其等电点为6.5，这与氨基酸分析预期的结果一致。化学组成最显著的特点是每个酶分子含有两个色氨酸、12个甲硫氨酸和四个半胱氨酸残基。尽管来自哺乳动物组织的磷酸甘油酸激酶彼此部分相似，但在丝氨酸、谷氨酸、甘氨酸、半胱氨酸、缬氨酸、亮氨酸、酪氨酸、色氨酸和精氨酸含量上发现了明显差异。对S-羧甲基化蛋白质的胰蛋白酶消化产物进行指纹图谱分析和柱色谱分析，证实了氨基酸分析的数据。当用对氯汞苯甲酸或5,5'-二硫代双（2-硝基苯甲酸）（Nbs2）进行修饰时，肝磷酸甘油酸激酶会失活。在变性条件下，该酶有两个可与Nbs2反应的巯基，其中一个对催化作用至关重要。用NaBH4还原后，用Nbs2测定每个分子有四个半胱氨酸残基，这表明存在一个二硫键。通过沉降平衡研究，发现分子量为49600。凝胶过滤得到的值为43000 - 50000。通过分析十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计分子量为45600。这与在6 M盐酸胍中通过薄层凝胶色谱得到的37500的值不一致。沉降速度实验表明沉降系数s20,w等于3.4 S。斯托克斯半径为2.77 nm，比容v为0.747 ml×g(-1)。通过分析凝胶过滤发现扩散系数为76.9 μm2×s(-1)。根据这些数据计算出分子量为44000。牛肝磷酸甘油酸激酶的其他物理常数为：摩擦比f/f0等于1.18，轴比等于3.3，最大水合度为每克蛋白质0.1克。用能使各种其他蛋白质解离的处理方法（8 M尿素、6 M盐酸胍、十二烷基硫酸钠、羧甲基化、马来酰化）处理，牛肝磷酸甘油酸激酶不能解离成更小的亚基。所有实验都有力地支持了该酶缺乏亚基结构的观点。将牛肝磷酸甘油酸激酶的一些特性与来自兔肌肉、酵母和人红细胞的相应蛋白质进行了比较。
