Lost organization of urate degrading enzymes in peroxisomes during animal evolution

嘌呤降解的终产物因物种而异。嘌呤降解的最终产物在人类、类人猿灵长类动物和新大陆猴中是尿酸盐，在除灵长类动物和旧大陆猴之外的哺乳动物中是通过尿酸酶的尿囊素，在一些硬骨鱼中是通过尿囊素酶（ALN）的尿囊酸，在鱼类和两栖动物中是通过尿囊素酶（ALC）的尿素，在许多无脊椎动物中是通过尿素酶的氨。而尿酸盐的降解在高等动物中不完全得多。我们曾报道，在海洋鱼类中，嘌呤降解为尿酸盐的酶位于细胞质中，而尿酸盐降解为尿素的酶位于过氧化物酶体中。这表明在嘌呤降解过程中，过氧化物酶体酶在进化过程中已经丢失。ALN和ALC是鱼类肝脏中不同的蛋白质，而两栖类肝脏中这两种酶形成复合物，我们研究了两栖类尿囊素酶-尿囊酸酶复合物（ALNC）的形成机制是如何丧失的 ...更多信息 （1）对两栖类ALNC亚基ALN的cDNA序列进行了测定。用牛蛙抗ALN抗体从λ gt 11文库中获得了一些克隆（2）。在海水鱼肝中，嘌呤降解的最终产物是尿素和乙醛酸。乙醛酸可以通过丙氨酸：乙醛酸氨基转移酶转化为甘氨酸，以重新利用嘌呤碳（3）。一般认为，所有的尿囊素降解酶在哺乳动物进化过程中都丢失了。令人惊讶的是，脲基乙醇酸裂解酶已在哺乳动物组织中发现。大鼠脲基乙醇酸裂解酶的表观Km（17 mM）远高于鱼肝脲基乙醇酸裂解酶的表观Km（0.33mM）。哺乳动物在进化过程中由于脲基乙醇酸的Km升高而丧失了体内功能（4）。我们报道了从λ gt 11中克隆的肝脏文库中分离编码青蛙过氧化氢酶的cDNA。从核苷酸序列推导出的528个氨基酸序列具有计算的Mr为59871。我们发现游离ALN仅具有ALN活性，与ALNC所组成的ALN亚基不同。酶活性表现为鱼类ALN和两栖类ALNC的中间型（5）。我们将鱼类分为咸水、半咸水和淡水三种生境，以及肉食性、草食性和杂食性三种食性。我们确定了ALN，过氧化物酶体基质或胞质溶胶的亚细胞分布。我们确定了ALC类型，过氧化物酶体基质或过氧化物酶体外表面的膜结合。用RT-PCR方法从mRNA中合成了各鱼的cDNA，并通过DNA测序仪测定了全长。我们将结果与蛙ALN亚基和爪蟾肝ALC的C-端氨基酸序列进行了比较，并确定了在过氧化物酶体中的转移和分布信号。

End products of purine degradation vary from species to species. The end product of purine degrade is urate in humans, hominoid primates and new world monkeys, allantoin by uricase in mammals other than primates and old world monkeys, allantoiate by allantoinase(ALN) in some teleosts, urea by allantoicase(ALC) 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. ALN and ALC are different proteins in fish liver, whereas the two enzymes form a complex in amphibian liver.We examined how the mechanism to form amphibian allantoinase-allantoicase complex(ALNC) is lost … More in higher animals during animal evolution (1).The cDNA encoding ALN of the subunit of amphibian ALNC was sequenced. We got some clones from λ gt11 library using anti ALN 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. Surprisingly, ureidoglycollate lyase has been found in mammalian tissue. The apparent Km(17mM) of the rat enzyme for ureidoglycollate was much higher than that (0.33mM)of fish-liver ureidoglycollate lyase. Mammals have lost the function in vivo by elevating the Km for ureidoglycollate during evolution (4).We reported the isolation of cDNA encoding frog catalase from a liver library cloned in λgt11. The 528 amino acid sequence deduced from the nucleotid sequence has a calculated Mr of 59871. 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).We separated the fish with three kinds of habitat : salt water, brackish water and fresh water and with three kinds of food habit : carnivorous,herbivorous and omnivovous. We determined the subcellular distribution of ALN, peroxisomes matrix or cytosoles. We determined ALC type, peroxisomal matrix or membrane-bound of outer surface of peroxisomes. The cDNA of each fish, was synthesized with RT-PCR using mRNA and The full-length was determined by the DNA sequencer. We compared the results with subunit of frog ALN and Xenopus liver ALC of C-end amino acid sequence and determined the signal of transference to and from distribution in peroxisomes Less