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G A Piazza

Publications and source records attributed to G A Piazza.

22 records · Page 2Linked to original sources
PubMed1989

Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection.

Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.

Animals↗
PubMed1989

Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix.

Dipeptidyl peptidase IV (DPP IV) is a cell surface glycoprotein which has been implicated in hepatocyte-extracellular matrix interactions [Hixson, DeLourdes, Ponce, Allison & Walborg (1984) Exp. Cell Res. 152, 402-414; Walborg, Tsuchida, Weeden, Thomas, Barrick, McEntire, Allison & Hixson (1985) Exp. Cell Res. 158, 509-518; Hanski, Huhle & Reutter (1985) Biol. Chem. Hoppe-Seyler 366, 1169-1176]. However, its proteolytic substrate(s) and/or binding protein(s) which mediate this influence have not been conclusively identified. Nitrocellulose binding assays using 125I-labelled DPP IV that was purified to homogeneity from rat hepatocytes revealed a direct interaction of DPP IV with fibronectin. Although fibronectin could mediate an indirect binding of DPP IV to collagen, no evidence was found for a direct binding of DPP IV to native or denatured Type I collagen. Fibronectin appeared to bind DPP IV at a site distinct from its exopeptidase substrate recognition site since protease inhibitors such as competitive peptide substrates and phenylmethanesulphonyl fluoride enhanced binding, possibly as a result of an altered conformation of DPP IV. To determine if fibronectin binding to DPP IV is involved in the interaction of fibronectin with the hepatocyte surface, the effect of various DPP IV inhibitors on 125I-fibronectin binding to isolated hepatocytes in suspension was examined. Kinetic studies revealed that inhibitors of DPP IV which enhanced fibronectin binding in vitro accelerated the initial binding of fibronectin to the cell surface where it was subsequently cross-linked (presumably by tissue transglutaminase) to as yet undefined components. Immunolocalization of fibronectin and DPP IV in normal rat liver sections showed that both proteins were present along the hepatocyte sinusoidal membrane. These observations, coupled with previous results showing that DPP IV is tightly bound to biomatrix isolated from rat liver (Hixson et al., 1984; Walborg et al., 1985), suggest that DPP IV binding to fibronectin may play a role in interactions of hepatocytes with extracellular matrix in vivo and possibly in matrix assembly.

Animals↗
PubMed1985

Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.

Calmodulin stimulated the rate of Mg2+-induced polymerization of human platelet actin. The stimulatory effect was due to an increase in the nucleation phase of the reaction; there was no effect on the steady-state viscosity. The calmodulin antagonist trifluoperazine blocked the stimulatory effect of calmodulin. Addition of EGTA to the reaction mixture also stimulated the rate of actin polymerization; however, the effect of calmodulin on actin polymerization is not due to Ca2+ chelation, as is presumed to be the case for EGTA. Electron microscopy revealed structural differences in the filaments prepared in the presence of calmodulin as compared to those prepared with trifluoperazine. In the presence of calmodulin, the filaments were thicker, suggesting that they consisted of multiple actin polymers. In addition, numerous projections were present perpendicular to the filaments, as well as localized areas of filament bundling. It was not possible to demonstrate a direct interaction between calmodulin and actin, which raises the possibility that the calmodulin effect may be indirect through a calmodulin-binding protein or calmodulin-dependent enzyme. Regardless of whether calmodulin is acting directly or indirectly, these results provide evidence that calmodulin may play a regulatory role in either the polymerization of actin or in determining the structural characteristics of actin filaments.

Actins↗