Johnson M D, Anderson B D
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City 84112, USA.
Pharm Res. 1996 Dec;13(12):1881-6. doi: 10.1023/a:1016001712524.
The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (Km and Vmax) of purified calf spleen PNP for inosine and 2',3'-dideoxyinosine (ddI) and of purified calf intestinal ADA for 2',3'-dideoxyadenosine (ddA), 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), and 2'-beta-fluoro-2', 3'-dideoxyadenosine (F-ddA) have been explored.
BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, gamma-glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest.
The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex.
ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddI, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.
测定牛脑微血管内皮细胞（BBMECs）、全脑组织和红细胞中嘌呤和嘧啶代谢酶嘌呤核苷磷酸化酶（PNP）、腺苷脱氨酶（ADA）和胞苷脱氨酶（CDA）的比活性。此外，还研究了纯化的小牛脾脏PNP对肌苷和2'，3'-双脱氧肌苷（ddI）以及纯化的小牛肠ADA对2'，3'-双脱氧腺苷（ddA）、6-氯-2'，3'-双脱氧嘌呤（6-Cl-ddP）和2'-β-氟-2'，3'-双脱氧腺苷（F-ddA）的底物特异性（Km和Vmax）。
通过两步酶分散处理从牛大脑皮层分离BBMECs，然后在50% Percoll密度梯度上进行离心。测定各种组织匀浆（大脑皮层、BBMECs和红细胞）中碱性磷酸酶、γ-谷氨酰转肽酶、ADA、PNP和CDA的活性。还使用市售酶和几种感兴趣的核苷类似物进行酶动力学研究。
大脑微血管中ADA和PNP的活性分别比大脑皮层高42倍和247倍，而微血管部分未检测到CDA活性，皮层中的总体活性也非常低。
ADA和PNP可能作为某些抗HIV双脱氧核苷的酶性血脑屏障。对ddI、ddA、6-Cl-ddP和F-ddA的脑内可及性模拟表明，酶定位的定量意义可能有很大差异，然而，这取决于药物的膜通透性及其在内皮细胞内的生物转化速率常数。
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