Lost organization of purine degrading enzymes in peroxisomes during animal evolution

嘌呤降解的最终产物因物种而异。嘌呤降解的最终产物在人类、类人猿和新大陆猴中为尿酸，在除灵长类动物和旧大陆猴以外的哺乳动物中为尿囊素，在某些硬骨鱼中为尿囊素酶，在鱼类和两栖动物中为尿囊素酶，在许多无脊椎动物中为氨。嘌呤降解为尿酸盐对所有动物物种都是常见的，而尿酸盐的降解在高等动物中要不完全得多。我们报道过，在海洋鱼类中，嘌呤到尿酸盐的降解酶位于细胞质中，而尿酸盐到尿素的降解酶位于过氧化物酶体中。这表明在嘌呤降解过程中，过氧化物酶在进化过程中丢失了。尿囊酶（Allantoinas， ALN）和尿囊酶（allantoicase， ALC）在鱼类肝脏中是两种不同的蛋白，而在两栖动物肝脏中则形成一种复合物。我们研究了两栖动物尿囊酶（allantoinase, allantoicase）在进化过程中丢失的机制。(1)测定了两栖动物ALNC亚基尿囊酶的cDNA序列。利用牛蛙抗尿囊酶从λat 11文库中获得了一些克隆。(2)在咸水鱼肝脏中，嘌呤降解的最终产物为尿素和乙醛酸盐。乙醛酸盐可以通过丙氨酸：乙醛酸氨基转移酶转化为甘氨酸，以再利用嘌呤碳。(3)一般认为，在哺乳动物的进化过程中，所有的尿囊素降解酶都丢失了。令人惊讶的是，在哺乳动物组织中发现了糖醛酸水解酶。大鼠溶脲醇酯酶的表观Km （17 mM）远高于鱼肝溶脲醇酯酶的表观Km （0.33 mM）。哺乳动物在进化过程中通过提高尿醛酸酯的Km而丧失了在体内的功能。(4)游离ALN仅具有ALN活性，与ALNC组成的ALN亚基不同。该酶表现为鱼类ALN和两栖动物ALNC的中间类型。(5)分别从沙丁鱼（咸水）、鲻鱼（咸水）和鲫鱼（淡水）的肝脏中纯化ALN和ALC。用蛋白测序仪对纯化的ALN和ALC部分进行氨基酸序列测定。利用mRNA进行RT-PCR合成各鱼的cDNA，用DNA测序仪测定其全长。在不同的生境中，每一种鱼的ALN和ALC的DNA序列信号向过氧化物酶体的转移和分布。我们比较了鱼类ALN和两栖动物ALNC的亚基。少

End products of purine degradation very from species to species. The end product of purine degradation is urate in humans, hominoid primates and new world monkeys, allantoin by uricase in mammals other than primates and old world monkeys, allantoate by allantoinase in some teleosts, urea by allantoicase in fish and amphibians and ammonia by urease in many invertebrates.The degradation of purines to urate is common to all animal species, while the degradation of urate is much less complete in higher animals. We have reported that in marine fish, the degrading enzymes of purines to urate are located in the cytosol, while the degrading enzymes of urate to urea are located in the peroxisomes. This shows that in purine degradation, peroxisomal enzymes have been lost during evolution. Allantoinas (ALN) and allantoicase (ALC) are different protein in fish liver, whereas the two enzymes form a complex in amphibian liver.We examined how the mechanism to form amphibian allantoinase-allantoicase … More complex (ALNC) is lost in higher animals during animal evolution.(1) The cDNA encoding allantoinase of the subunit of amphibian ALNC was sequenced. We got some clones from λat 11 library using anti allantoinase of bullfrog. (2) In saltwater fish liver, the end products of purine degradation were urea and glyoxylate. Glyoxylate may be converted to glycine by alanine : glyoxylate aminotransferase for the reutilization of purine carbons. (3) It is generally accepted that all of the allantoin-degrading enzymes were lost during mammalian evolution. Suprisingly, ureidoglycollate lyase has been found in a mammalian tissue. The apparent Km (17 mM) of the rat enzyme for ureidoglycollate was much higher than that (0.33 mM) of fish-liver ureidoglycollate lyase. Mammals have lost the function in vivo by elevating the Km for ureidoglycollate during evolution. (4) We found free ALN only with ALN activity which is differ from ALN subunit composed of ALNC. The enzyme shows the middle type of fish ALN and amphibian ALNC. (5) ALN and ALC were purified from the each liver of sardine (salt water), mullet (brackish water) and crucian carp (fresh water). Amino acid sequences were determined from the part of purified ALN and ALC by protein sequencer. The cDNA of each fish was synthesied with RT-PCR using mRNA and determined full-length by DNA sequencer. In defferent habitat, DNA sequence of each fish ALN and ALC signal of transference to and destribution in peroxisomes. We compared with subunit of fish ALN and amphibian ALNC. Less