Isolation and characterization of calmodulin from an insulin-secreting tumour

Abstract

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.

Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells

“…Tissues. Insulinomas were propagated in New England Deaconess Hospital (NEDH) rats as described previously [14]. Tumours were removed and tissue was scraped from the fibrous capsules into Hanks saline [(in retool/l) 137 NaC1, 5.4 KC1, 1.67 MgSO 4, 4 CaC12, 0.34 NazHPO 4 and 4.2 NaHCO 3 (pH 7.4)].…”

Ca2+—calmodulin dependent myosin light‐chain phosphorylating activity in insulin‐secreting tissues

“…The rat islet cell tumour was propagated by subcutaneous implantation in an inbred strain of rats [9,19]. Pancreatic islets were isolated by collagenase digestion [20] from untreated animals of the same strain.…”

“…Samples (2-50 c(g protein) were incubated for I-20 mm at 30°C in 50 /.d 50 mM MES (pH 6.9) containing 1 mM MgC12, 0.1 mM DTT, 5.1 mM EGTA and 60 PM [Y-~~P] ATP (4 X lo6 dpm; prepared as in [21] from carrier free 32Pi; Amersham Intl., Bucks.). Other additions included 5 /.IM chicken gizzard MLC (prepared as in [22]), 1.2 PM bovine brain calmodulin (prepared as in [9]) and 5 mM CaC12 which resulted in free Ca2+ estimated at 15 PM [23]. The reaction was terminated by addition of 30 /.d 200 mM TrisHCl (pH 6.8) containing 20% (w/v) sucrose, 2% (w/v) SDS, 10 mM DTT, 0.02% bromophenol blue and heating to 1OO'C for 5 min.…”

“…The key regulatory component in this process may be an increase in the intracellular free [Ca"] (review [7]) which would promote Ca"/calmodulin interaction. Insulin secreting tissues have a high content of calmoduhn [8,9] and insulin secretion is inhibited by calmodulin antagonists [ 1 O-l 21.…”

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules

“…B. Frank (Eli Lilly, Indianapolis, IN, U.S.A.). Ala-LysArg-CH2Cl and Ala-Nle-Arg-CH2Cl were kindly provided by Dr. E. Shaw (Friedrich Miescher-Institut, (Hutton et al, 1981). Insulin-secretory granules were.…”