McDevitt T M, Todorov P T, Beck S A, Khan S H, Tisdale M J
Pharmaceutical Sciences Institute, Aston University, Birmingham, United Kingdom.
Cancer Res. 1995 Apr 1;55(7):1458-63.
A scheme is described for the purification of a lipid-mobilizing factor from a cachexia-inducing murine tumor (MAC16) using a combination of ion exchange (Mono Q), exclusion (Superose), and hydrophobic (C8) chromatography. This process yields an active material with an apparent molecular weight of 24,000 with an overall purification of 3,500 from the tumor homogenate and representing 0.005% of the total protein present. The material tends to aggregate to high molecular mass, is acidic (pI < 4), and displays heterogeneity of charge as evidenced by a broad elution profile on ion exchange and exclusion chromatography and multiple peaks on hydrophobic columns. The purified material was heat and alkali (pH 10.4) labile and activity could be completely inhibited by sulfatase, suggesting that the negative charge could arise from sulfate residues. There was no evidence that the material possessed triglyceride lipase activity. Animals transplanted with the MAC16 tumor and with a delayed weight loss contained in their serum antibodies that recognized a M(r) 24,000 band on Western blots. This material copurified with the lipid-mobilizing factor. Such antibodies were not present in the serum of mice transplanted with the MAC13 tumor, which does not induce cachexia, suggesting that the antibodies were directed to the induction of cachexia rather than the tumor itself. Urine from patients with cancer cachexia also contained a lipid-mobilizing factor which adhered to DEAE-cellulose and gave an apparent M(r) of 24,000 by exclusion chromatography. Western blotting using serum from MAC16 tumor-bearing animals showed the presence of a band of M(r) 24,000 in such fractions, which was not detected using serum from mice bearing the MAC13 tumor. This band was not present in Western blots of urine from normal subjects. The fact that serum from mice bearing the MAC16 tumor can detect the human lipid-mobilizing activity suggests a high degree of structural similarity between the two and raises the possibility that cachexia in humans may be caused by the same species as in the mouse.
本文描述了一种从诱导恶病质的小鼠肿瘤（MAC16）中纯化脂质动员因子的方案，该方案结合了离子交换（Mono Q）、排阻（Superose）和疏水（C8）色谱法。此过程产生了一种表观分子量为24,000的活性物质，从肿瘤匀浆中总体纯化了3500倍，占总蛋白的0.005%。该物质倾向于聚合成高分子量，呈酸性（pI < 4），并且电荷表现出异质性，这通过离子交换和排阻色谱上的宽洗脱谱以及疏水柱上的多个峰得以证明。纯化后的物质对热和碱（pH 10.4）不稳定，其活性可被硫酸酯酶完全抑制，这表明负电荷可能源于硫酸酯残基。没有证据表明该物质具有甘油三酯脂肪酶活性。移植了MAC16肿瘤且体重减轻延迟的动物血清中含有能识别Western印迹上M(r) 24,000条带的抗体。该物质与脂质动员因子共纯化。移植了不诱导恶病质的MAC13肿瘤的小鼠血清中不存在此类抗体，这表明这些抗体针对的是恶病质的诱导而非肿瘤本身。癌症恶病质患者的尿液中也含有一种脂质动员因子，它能与DEAE - 纤维素结合，通过排阻色谱法测得其表观M(r)为24,000。使用来自携带MAC16肿瘤动物的血清进行Western印迹分析表明，此类组分中存在M(r) 24,000的条带，而使用携带MAC13肿瘤小鼠的血清未检测到该条带。正常受试者尿液的Western印迹中不存在此条带。携带MAC16肿瘤小鼠的血清能够检测到人类脂质动员活性这一事实表明两者在结构上具有高度相似性，并增加了人类恶病质可能由与小鼠相同的物质引起的可能性。
