Criscuolo G R, Merrill M J, Oldfield E H
Surgical Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland.
J Neurosurg. 1988 Aug;69(2):254-62. doi: 10.3171/jns.1988.69.2.0254.
The nature of vascular permeability factor (VPF) activity derived from serum-free conditioned medium containing cultured human malignant glial tumors has been further investigated. A 1000-fold purification was accomplished by sequential heparin-Sepharose affinity chromatography and high-performance liquid chromatography gel filtration chromatography steps. Vascular permeability factor activity falls into a molecular weight range of 41,000 to 56,000 D. Activity is bound to hydroxylapatite, carboxymethyl-Sepharose, phenyl-Sepharose, and heparin-Sepharose, whereas little or no activity was bound to diethylaminoethyl-Sephacel. Vascular permeability factor activity is trypsin- and pepsin-sensitive but is unaffected by treatment with ribonuclease A. This suggests that VPF is a hydrophobic, positively charged (cationic) polypeptide with a potentially biologically significant affinity for heparin. As most proteins are negatively charged (anionic) and have no affinity for heparin, a significant advantage was gained by performing these purification steps. The activity of VPF is not inhibited by coinjection of conditioned medium with soybean trypsin inhibitor; or hexadimethrine (both known antagonists of tissue plasminogen activator, Hageman factor, and serum kallikrein); or aprotinin (an antagonist of both plasmin and tissue kallikrein); or phenylmethanesulfonyl fluoride (a serine esterase (elastase) inhibitor); or pepstatin-A (an acid protease inhibitor which inactivates vascular permeability-inducing leukokinins). These data, together with the fact that VPF is produced and released into serum-free media, provides substantial evidence against it being one of the more commonly known serum-derived permeability mediators. Treatment with dithiothreitol inhibited VPF activity, indicating the presence of at least one essential disulfide bond in this molecule. Inhibition by dexamethasone of VPF expression in cultured malignant glial cells appears to be selective. Dexamethasone-induced inhibition of VPF was dose-responsive and was not associated with a parallel inhibition of cellular protein synthesis as determined by tritiated leucine incorporation into trichloroacetic acid-precipitable material. Inclusion of dexamethasone in the culture medium was not associated with altered cell viability or cell number. A series of in vivo studies confirmed the inhibition of VPF activity in test animals pretreated with dexamethasone. This steroid-induced inhibition was partially reversed by treatment of test animals with actinomycin D prior to exposure to dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)
对源自含有培养的人类恶性胶质瘤的无血清条件培养基的血管通透性因子（VPF）活性的性质进行了进一步研究。通过依次进行肝素 - 琼脂糖亲和层析和高效液相色谱凝胶过滤层析步骤，实现了1000倍的纯化。血管通透性因子活性的分子量范围在41,000至56,000道尔顿之间。活性与羟基磷灰石、羧甲基 - 琼脂糖、苯基 - 琼脂糖和肝素 - 琼脂糖结合，而与二乙氨基乙基 - 琼脂糖几乎没有或没有结合。血管通透性因子活性对胰蛋白酶和胃蛋白酶敏感，但不受核糖核酸酶A处理的影响。这表明VPF是一种疏水性、带正电荷（阳离子）的多肽，对肝素有潜在的生物学显著亲和力。由于大多数蛋白质带负电荷（阴离子）且对肝素无亲和力，通过进行这些纯化步骤获得了显著优势。VPF的活性不会因将条件培养基与大豆胰蛋白酶抑制剂、己二甲铵（两者均为组织纤溶酶原激活剂、哈格曼因子和血清激肽释放酶的已知拮抗剂）、抑肽酶（纤溶酶和组织激肽释放酶的拮抗剂）、苯甲磺酰氟（一种丝氨酸酯酶（弹性蛋白酶）抑制剂）或胃蛋白酶抑制剂A（一种使诱导血管通透性的白细胞激肽失活的酸性蛋白酶抑制剂）共同注射而受到抑制。这些数据，连同VPF在无血清培养基中产生并释放这一事实，提供了大量证据证明它不是一种更常见的源自血清的通透性介质。用二硫苏糖醇处理可抑制VPF活性，表明该分子中至少存在一个必需的二硫键。地塞米松对培养的恶性胶质细胞中VPF表达的抑制似乎具有选择性。地塞米松诱导的VPF抑制呈剂量依赖性，并且与通过将氚标记的亮氨酸掺入三氯乙酸可沉淀物质所确定的细胞蛋白质合成的平行抑制无关。在培养基中加入地塞米松与细胞活力或细胞数量的改变无关。一系列体内研究证实了用地塞米松预处理的实验动物中VPF活性受到抑制。在用放线菌素D处理实验动物使其暴露于地塞米松之前，这种类固醇诱导的抑制作用部分得到逆转。（摘要截断于400字）
