The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase
The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase
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DOI：
10.1042/bj20031930
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发表时间：
2004-08-01
影响因子：
4.1
通讯作者：
Borst, DW
中科院分区：
文献类型：
--
作者：
Li, S;Friesen, JA;Borst, DW
在之前的一项研究中[Li、瓦格纳、弗里森和Borst（2003）Gen. Comp. Endocrinol. 134，147-155]，我们表明美洲龙虾的MO（下颌器官）具有高水平的HMGR（3-羟基-3-甲基戊二酰-CoA还原酶），并且大多数（约100 - 200）的MO（下颌器官）具有高水平的HMGR（3-羟基-3-甲基戊二酰-CoA还原酶）。75%）的酶活性是可溶的。在本研究中，我们报告了这种酶的生化和分子特征。HMGR在MO中有两种形式：更丰富的可溶性形式（66 kDa）和较少的膜结合形式（72 kDa）。从MO中分离出两个HMGR的cDNA。一个2.6 kb的cDNA编码HMGR 1，一个599个氨基酸的蛋白质（63 kDa），和一个3.2 kb的cDNA编码HMGR 2。655个氨基酸的蛋白质（69 kDa）。这两个cDNA具有相同的3 '末端，并且似乎是单个基因的产物。这两个蛋白质的推导的氨基酸序列揭示了高度的相似性，其他I类HMGRs。疏水图显示HMGR 1的N端缺少跨膜区，HMGR 2只有一个跨膜区。在Sf 9昆虫细胞中表达的重组HMGR 1是可溶的，并且具有与来自MO的天然HMGR相似的动力学特征。用磷酸酶处理不影响HMGR活性，这与HMGR 1和HMGR 2在位置490或546（在来自高等真核生物的I类HMGR中发现的保守磷酸化位点的位置）处均不具有丝氨酸的观察结果一致。其他龙虾组织（即中肠、脑和肌肉）的HMGR活性和mRNA水平较低。具有较高HMGR活性的MO具有较高的HMGR mRNA水平，这意味着HMGR部分在转录水平上受到调节。
In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75 %) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2. a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.