Kumar R, Prasad R
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, 160 012, Chandigarh, India.
Biochim Biophys Acta. 2000 Dec 20;1509(1-2):429-39. doi: 10.1016/s0005-2736(00)00325-4.
Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient (H greater than H). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.
从肾刷状缘膜（BBM）中纯化的主要锌结合蛋白（R. Kumar，R. Prasad，《生物化学与生物物理学报》1419 (1999) 23）被重构到脂质体中，并对其功能特性进行了研究。通过SDS-PAGE检查主要锌结合蛋白在蛋白脂质体中的物理掺入情况，银染显示为单一条带。通过相差显微镜评估蛋白脂质体的结构完整性，结果显示蛋白脂质体为球状结构且边界完整。通过监测在2 mM Ca(2+)、Cd(2+)或Zn(2+)存在下预加载的蛋白脂质体中Zn(2+)的流出，进一步检查蛋白脂质体的结构完整性/渗漏情况。观察到即使在流出开始2小时后，85 - 95%的Zn(2+)仍保留在蛋白脂质体中，这表明蛋白脂质体在摄取研究期间没有渗漏并保持了结构完整性。锌进入蛋白脂质体遵循米氏动力学，亲和常数（K(m)）为1.03 mM，最大速度（V(max)）为每分钟1333 nmol/mg蛋白。摄取过程遵循一级动力学，速率常数（k）为1.09×10(-3) s(-1)。通过研究二价阳离子即Ca(2+)和Cd(2+)与锌摄取的相互作用来确定锌转运系统的特异性。观察到Cd(2+)竞争性抑制锌摄取过程，抑制浓度（K(i)）为2.9 mM。Cd(2+)对锌摄取抑制作用的动力学分析表明K(m)增加到1.74 mM，而不影响V(max)。发现锌进入蛋白脂质体对温度敏感，阿累尼乌斯图在27℃处出现转折点。在转折点以下和以上，表观活化能（E(a)）分别为7.09和2.74 kcal/mol。锌摄取的初始速度随着外向质子梯度（H大于H）的增加而增加。锌摄取受到二环己基碳二亚胺（DCCD）的抑制，这表明-COOH基团参与了锌跨脂质双层的转运。酸性氨基酸与碱性氨基酸的比例（1.26）强烈表明它是一种酸性蛋白。该蛋白中的半胱氨酸含量微不足道，这进一步证实了酸性氨基酸可能是与锌结合的主要候选者的可能性。本研究结果证实，从肾BBM中纯化的40 kDa主要锌结合糖蛋白是一种参与锌流入肾小管系统上皮细胞的锌转运蛋白。
