Lost organization of urate degrading enzymes in peroxisomes during animal evolution

嘌呤降解的最终产物因物种不同而不同。在人类、类人灵长类动物和新大陆猴子中，嘌呤降解的最终产物是尿酸，在灵长类和东半球猴子以外的哺乳动物中是尿囊素，在一些硬骨动物中是尿囊素酶(ALN)，在鱼类和两栖动物中是尿囊酸酶(ALC)尿素，在许多无脊椎动物中是尿素酶(ALC)。我们曾报道，在海洋鱼类中，嘌呤降解为尿酸的酶位于胞浆中，而尿酸降解为尿素的酶位于过氧化体中。这表明，在嘌呤降解过程中，过氧化物酶在进化过程中已经丢失。Aln和Alc是鱼肝脏中不同的蛋白质，而这两种酶在两栖动物肝脏中形成一个复合体。我们研究了两栖尿囊素酶-尿囊酸酶复合体的形成机制是如何丢失的…在高等动物进化过程中更多(1)。对两栖动物ALNC亚基编码ALN的基因进行了测序。利用牛蛙抗碱性蛋白(2)从λgt11文库中获得了一些克隆。在咸水鱼肝脏中，嘌呤降解的最终产物是尿素和乙醛。丙氨酸：乙醛酸氨基转移酶可将乙醛酸转化为甘氨酸，以重新利用嘌呤碳(3)。人们普遍认为，在哺乳动物进化过程中，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尿苷乙醇酸裂解酶。大鼠尿素乙二酸酯酶的表观Km(17 MM)远高于鱼肝尿素乙二酸酯酶的表观Km(0.33 mM)。哺乳动物已经在体内失去了这种功能，因为在进化过程中脲乙醇酸的Km升高(4)。我们报道了从λgt11克隆的肝脏文库中分离到编码蛙过氧化氢酶的cDNA.从核苷酸序列推导出的528个氨基酸序列的计算MR为59871。我们发现，只有游离的ALN具有ALN活性，这与由ALNC组成的ALN亚基不同。该酶表现为中间类型的鱼类ALN和两栖动物ALNC(5)。我们将鱼类分为三种生境：咸水、微咸水和淡水，并分别具有三种食性：肉食性、草食性和杂食性。我们测定了ALN、过氧化物酶体基质或胞浆的亚细胞分布。我们测定了ALC类型、过氧化体基质或过氧化体外表面的膜结合。用RT-PCR方法合成每条鱼的cDNA，并用DNA测序仪测定其全长。我们将结果与蛙ALN和非洲爪哇肝脏ALC的C端氨基酸序列进行了比较，并确定了传递信号在过氧化体中分布较少

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