Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration.

Inter-alpha-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.

Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.

Selective inactivation of butyrylcholinesterase with metal chelators suggests there is more than one metal binding site.

Cholinesterases exhibit functions apart from their esterase activity. We have demonstrated an aryl acylamidase and a zinc stimulated metallocarboxypeptidase activity in human serum butyrylcholinesterase. To establish the presence of zinc binding sites in the enzyme we examined the effect of metal chelators on its catalytic activities. The metal chelators 1,10-phenanthroline and N,N,N',N'-tetrakis (2-pyridyl methyl)ethylene diamine (TPEN) inhibited all the three catalytic activities in the enzyme. However, EDTA inhibited the peptidase activity exclusively without affecting the cholinesterase and aryl acylamidase activities. The catalytic activities were recovered upon removal of the chelator by Sephadex G-25 chromatography. Pre-treatment of the enzyme with any one of the three chelators resulted in the binding of the enzyme to a zinc-Sepharose column or to 65Zn2+. Histidine modification of the enzyme pretreated with chelators resulted in abolition of 65Zn2+ binding and zinc-Sepharose binding. Whereas the binding studies demonstrated removal of a metal from a Zn2+ binding site, attempts to remove the metal responsible for catalytic activity were unsuccessful. Atomic absorption spectroscopy indicated approximately 2.5 mol of zinc per mol of enzyme before treatment with EDTA and 1 mol zinc per mol enzyme after EDTA treatment. The results indicate that there are at least two metal binding sites on butyrycholinesterase. The presence of two HXXE...H sequences in butyrylcholinesterase supports these findings. Our studies implicate a zinc dependent metallocarboxypeptidase activity in the non-cholinergic functions of butyrylcholinesterase.

Evidence for a Zn(2+)-binding site in human serum butyrylcholinesterase.

Purified human serum butyrylcholinesterase after treatment with either of the metal chelators EDTA or NaCN was able to bind to a Zn(2+)-chelate-Sepharose affinity column and was eluted from the column by EDTA or imidazole. Prior EDTA treatment of the enzyme was essential for binding to this affinity column. The enzyme could be labelled with (65)Zn(2+) after EDTA treatment of the enzyme. Diethylpyrocarbonate modification of histidine residues in the EDTA-treated enzyme resulted in the abolition of both binding to the Zn(2+)-chelate-Sepharose column and labelling by (65)Zn(2+). Stoicheiometry of (65)Zn(2+) binding indicated approximately 0.85 mol of Zn(2+)/mol of subunit of the EDTA-treated enzyme. EDTA or NaCN treatment resulted in the loss of thermal stability of the enzyme at 37 degrees C which could not be reversed by Zn(2+). Whereas the cholinesterase activity of butyrlcholinesterase was not affected by EDTA, there was significant loss of its carboxypeptidase activity in the presence of EDTA, and the loss could be reversed by added ZnCl2. These results suggest the presence of a Zn(2+)-binding site on human serum butyrylcholinesterase and the involvement of histidine residues in the metal binding. The presence in human serum butyrylcholinesterase of a sequence HXXE...H found in many known Zn(2+)-containing enzymes supports these findings.

Noncholinergic functions of cholinesterases.

Cholinesterases (acetylcholinesterase and butyrylcholinesterase) exhibit additional catalytic activities apart from their well-known action in hydrolyzing choline esters. An amine-sensitive aryl acylamidase activity is exhibited by both acetyl- and butyrylcholinesterases. A metallocarboxypeptidase-like activity is found associated with both acetyl- and butyrylcholinesterases. The peptidase activity exhibited by butyrylcholinesterase was located in a 50-kDa COOH-terminal fragment. Acetylcholinesterase is implicated in noncholinergic functions in the substantia nigra. A relationship between tumorigenesis, cell differentiation, and cholinesterases has been speculated. The sequence similarities between different esterases, lipases, thyroglobulin, cell adhesion proteins, and cholinesterases would make it appear that cholinesterases are capable of exhibiting more than one biological activity and their functions are wider than what is hitherto known.