“…Western blot analysis of PMR-1 following estrogen stimulation+ Ten microgram samples of hepatocyte protein isolated at the indicated times following estrogen stimulation were analyzed by Western blot using a polyclonal antiserum to PMR-1+ Lane 1 is a standard of 1 ng of purified PMR-1+ A parallel blot of 100 ng of hepatocyte protein per lane was probed with sheep antiserum to vitellogenin as a control for estrogen stimulation+ ern blot in Figure 1 provide suggestive evidence that, like hMPO, PMR-1 is made as a larger precursor and is proteolytically processed to the form identified on polysomes+ It may or may not be a coincidence that the site of this putative processing event occurs at a position similar to that which separates the propeptide and light chain of hMPO+ Expression of the full-length PMR-1 cDNA in a recombinant baculovirus (Fig+ 4) yielded the 80-kDa protein (PMR80) expected from the sequence, but this was not processed to the 60-kDa form isolated from liver polysomes and lacked enzymatic activity+ These data indicate both that Sf9 cells lack the enzymatic pathway by which PMR-1 is proteolytically processed, and that processing is required to generate a catalytically active form of the enzyme+ In contrast, the recombinant 60-kDa form of PMR-1 estimated to represent the protein isolated from polysomes (PMR60) is an active endonuclease (Figs+ 5-7)+ PMR-1 was originally identified by its ability to generate a doublet cleavage product from a portion of the 59 end of albumin mRNA (Pastori et al+, 1991b)+ This was later mapped to consensus overlapping APyrUGA elements in a singlestranded loop region (Chernokalskaya et al+, 1997)+ That study also demonstrated that the purified enzyme also cleaved albumin mRNA at a number of sites that did not correspond to this consensus sequence+ Recombinant PMR60 did not cleave albumin mRNA at consensus APyrUGA elements (Fig+ 7)+ However, the recombinant enzyme generated much the same pattern of nonconsensus cleavages as the purified enzyme, indicating both that it is an endonuclease and that it is indeed PMR-1+ The reason recombinant PMR60 does not cleave at APyrUGA elements is not known, although we suspect it is related to folding of the protein, because a similar result was obtained with purified PMR-1 that was renatured after treatment under denaturing conditions (data not shown)+ It is unclear how a putative messenger RNase evolved from the peroxidase gene family+ Myeloperoxidase lacks detectable ribonuclease activity (Fig+ 2C), indicating this family of proteins does not have a secondary enzymatic function+ Perhaps some insight into this may be found in the dual function of several metabolic enzymes as RNA-binding proteins+ IRP-1 functions as a regulator of iron metabolism through its role in the translational repression of ferritin mRNA (Gray & Hentze, 1994) and stabilization of transferrin receptor mRNA (Binder et al+, 1994)+ Iron binding to the active site of this protein converts IRP1 from its RNA-binding form to cytoplasmic aconitase (Hirling et al+, 1994)+ Glyceraldehyde-3-phosphosphate dehydrogenase is a well-characterized hydrolytic enzyme which can bind AU-rich RNA sequence elements through its NAD(ϩ) binding site (Nagy & Rigby, 1995)+ Glutamate dehydrogenase was identified as the protein whose binding to the 39 UTR of cytochrome c oxidase mRNA correlates with liver-specific mRNA stabilization (Preiss et al+, 1995), and AUH, an ARE-binding protein, has enol...…”