Linden J, Prater M R, Sullivan G W, Johns R A, Patel A
Department of Internal Medicine (Cardiology), University of Virginia, Charlottesville 22908.
Biochem Pharmacol. 1991 Jan 15;41(2):273-9. doi: 10.1016/0006-2952(91)90486-o.
We have measured cyclic GMP accumulation in co-cultures of bovine aortic endothelial cells and rat smooth muscle cells as an index of endothelium-derived relaxing factor (EDRF) production. Adenosine deaminase (EC 3.5.4.4, Sigma type VI) produced a 5- to 10-fold increase in the basal and bradykinin-stimulated cyclic GMP content of co-cultures but had no effect on smooth muscle cells alone. Cyclic GMP accumulation in response to adenosine deaminase was not blocked by adenosine deaminase inhibitors or affected by adenosine, the products of adenosine deamination (inosine and ammonia), or adenosine receptor antagonists. Since superoxide anion is known to destroy EDRF and nitric oxide (NO) (which is similar or identical to EDRF in composition), we tested for superoxide dismutase (SOD, EC 1.15.1.1) in single lots of eight commercial sources of adenosine deaminase by measuring inhibition of the superoxide-mediated reduction of cytochrome c. SOD activity was found in all sources of adenosine deaminase, but varied widely. One lot of Sigma type VI enzyme contained 0.08 units SOD/unit adenosine deaminase. The EC50 values of purified SOD (0.23 units/mL) and Sigma type VI adenosine deaminase (2.1 units/mL) needed to increase the cyclic GMP content of co-cultures differed by a similar factor, 0.11. Thus, the SOD activity in adenosine deaminase is sufficient to account for its effect on cyclic GMP accumulation. One lot of Boehringer Mannheim adenosine deaminase contained much less SOD contamination (0.006 units SOD/unit adenosine deaminase) and produced much less accumulation of cyclic GMP in co-cultures. Cyclic GMP accumulations in response to adenosine deaminase and SOD were both abolished by the NO synthetase inhibitor NG-monomethyl-L-arginine (0.1 mM), consistent with the idea that these enzymes act by stabilizing EDRF. Adenosine deaminase and the SOD activity contaminating it were found to have similar molecular masses of 33-34 kD as assessed by gel permeation chromatography. When run under reducing conditions to dissociate homodimeric SOD into monomers, a 16.6 kD peptide which co-migrates with purified cupro-zinc SOD was visible in silver-stained sodium dodecyl sulfate-polyacrylamide gels of the Sigma type VI but not the Boehringer Mannheim adenosine deaminase. We conclude that commercial sources of adenosine deaminase are variably contaminated by SOD. Since EDRF is synthesized by many tissues, the use of adenosine deaminase contaminated with SOD may produce numerous effects not attributable to the deamination of adenosine.
我们已测定牛主动脉内皮细胞与大鼠平滑肌细胞共培养体系中环状鸟苷酸（cGMP）的积累情况，以此作为内皮源性舒张因子（EDRF）产生的指标。腺苷脱氨酶（EC 3.5.4.4，Sigma VI型）使共培养体系中基础状态及缓激肽刺激后的cGMP含量增加了5至10倍，但对单独的平滑肌细胞无影响。腺苷脱氨酶引起的cGMP积累不受腺苷脱氨酶抑制剂的阻断，也不受腺苷、腺苷脱氨产物（肌苷和氨）或腺苷受体拮抗剂的影响。由于已知超氧阴离子会破坏EDRF和一氧化氮（NO）（其组成与EDRF相似或相同），我们通过测量超氧阴离子介导的细胞色素c还原的抑制情况，对来自八个商业来源的单批腺苷脱氨酶进行了超氧化物歧化酶（SOD，EC 1.15.1.1）检测。在所有腺苷脱氨酶来源中均发现了SOD活性，但差异很大。一批Sigma VI型酶每单位腺苷脱氨酶含有0.08单位SOD。纯化的SOD（0.23单位/mL）和Sigma VI型腺苷脱氨酶（2.1单位/mL）增加共培养体系中cGMP含量所需的半数有效浓度（EC50）值相差类似倍数，为0.11。因此，腺苷脱氨酶中的SOD活性足以解释其对cGMP积累的影响。一批勃林格殷格翰腺苷脱氨酶的SOD污染要少得多（每单位腺苷脱氨酶含0.006单位SOD），并且在共培养体系中产生的cGMP积累也少得多。NO合酶抑制剂NG-单甲基-L-精氨酸（0.1 mM）消除了腺苷脱氨酶和SOD引起的cGMP积累，这与这些酶通过稳定EDRF起作用的观点一致。通过凝胶渗透色谱法评估，发现腺苷脱氨酶及其污染的SOD活性具有相似的分子量，为33 - 34 kD。在还原条件下运行以使同二聚体SOD解离为单体时，在Sigma VI型腺苷脱氨酶的银染十二烷基硫酸钠 - 聚丙烯酰胺凝胶中可见一条与纯化的铜锌SOD共迁移的16.6 kD肽段，而勃林格殷格翰腺苷脱氨酶中则没有。我们得出结论，商业来源的腺苷脱氨酶受到SOD的不同程度污染。由于许多组织都会合成EDRF，使用被SOD污染的腺苷脱氨酶可能会产生许多并非由腺苷脱氨引起的效应。
