Dataset Information

Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

ABSTRACT:

Unlabelled

The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.

Background

GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.

Methodology/principal findings

From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.

Conclusion/significance

Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

SUBMITTER: Hnia K

PROVIDER: S-EPMC2588339 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Publications

Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

Hnia Karim K Notarnicola Cécile C de Santa Barbara Pascal P Hugon Gérald G Rivier François F Laoudj-Chenivesse Dalila D Mornet Dominique D

PloS one 20081205 12

<h4>Unlabelled</h4>The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractil ...[more]

PMID: 19057650

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Project description:Caldesmon, an actin- and calmodulin-binding protein of smooth muscle, is a protein serine/threonine kinase capable of Ca2+/calmodulin-dependent autophosphorylation [Scott-Woo & Walsh (1988) Biochem. J. 252, 463-472]. Phosphorylation nullifies the inhibitory effect of caldesmon on the actin-activated Mg2+-ATPase activity of smooth-muscle myosin [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have characterized the kinase activity of caldesmon of chicken gizzard smooth muscle. Autophosphorylation requires Ca2+/calmodulin, but is unaffected by other second messengers (Ca2+/phospholipid/diacylglycerol, cyclic AMP or cyclic GMP), and is inhibited by the calmodulin antagonists chlorpromazine and compound 48/80, with 50% inhibition at 39.8 microM and 12.0 ng/ml respectively. Half-maximal activation of autophosphorylation occurs at 60-80 nM-Ca2+ and 0.14 microM-calmodulin, and maximal activity at 0.14-0.18 microM-Ca2+ and 1 microM-calmodulin; activation is gradually lost at higher Ca2+ and calmodulin concentrations. Autophosphorylation is pH-dependent, with maximal activity over the range pH 7-9, and requires free Mg2+ in addition to the MgATP2- substrate. The Km for ATP is 15.6 +/- 4.1 microM (mean +/- S.D., n = 4), and kinase activity is inhibited by increasing ionic strength [half-maximal inhibition at I = 0.094 +/- 0.009 M (mean +/- S.D., n = 4)]. Autophosphorylation does not affect the rate of hydrolysis of caldesmon (free or bound to calmodulin) by alpha-chymotrypsin. However, a slight difference in peptides generated from phospho- and dephospho-forms of caldesmon is observed. The binding of phospho- or dephospho-caldesmon to F-actin protects the protein against chymotryptic digestion, but does not alter the pattern of peptide generation. Characterization of proteolytic fragments of caldesmon generated by alpha-chymotrypsin and Staphylococcus aureus V8 protease enables localization of the phosphorylation sites and the kinase active site within the caldesmon molecule.

Project description:The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (∼5.7/min) similar to that of SPCs (∼4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ∼3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ∼20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.

Dataset Information

Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

Unlabelled

Background

Methodology/principal findings

Conclusion/significance

Publications

Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

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