Lost organization of urate 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酶（ALN）降解尿囊素，在鱼类和两栖动物中为尿囊酶（ALC）降解尿素，在许多无脊椎动物中为脲酶降解氨。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。ALN和ALC在鱼类肝脏中是不同的蛋白质，而这两种酶在两栖动物肝脏中形成复合物。我们研究了两栖动物尿囊酶-尿囊酶复合物（allantoicase complex， ALNC）的形成机制是如何在动物进化过程中丢失的。对两栖动物ALNC亚基编码ALN的cDNA进行了测序。利用牛蛙的抗ALN从λ gt11文库中获得了一些克隆(2)。在咸水鱼的肝脏中，嘌呤降解的最终产物是尿素和乙醛酸盐。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丢失。令人惊讶的是，在哺乳动物组织中发现了解糖酶。大鼠溶脲醇酯酶的表观Km（17mM）远高于鱼肝溶脲醇酯酶的表观Km（0.33mM）。哺乳动物在进化过程中通过提高尿苷酸的Km而失去了体内功能(4)。我们报道了从λgt11克隆的青蛙肝脏文库中分离到编码过氧化氢酶的cDNA。由核基序列推导出的528个氨基酸序列的Mr值为59871。我们发现游离ALN只具有ALN活性，与由ALNC组成的ALN亚基不同。该酶为鱼类ALN和两栖动物ALNC的中间类型(5)。我们将鱼分为三种栖息地：咸水、微咸水和淡水，以及三种饮食习惯：食肉、食草和杂食。我们测定了ALN、过氧化物酶体基质或细胞质的亚细胞分布。测定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。利用mRNA进行RT-PCR合成各鱼的cDNA，并利用DNA测序仪测定其全长。我们将结果与青蛙ALN和爪蟾肝ALC的c端氨基酸序列亚基进行了比较，并确定了在过氧化物酶体中分布的转移信号

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