Selected bovine eye tissues (cornea, lens, iris, ciliary body,
vitreous body, retina, choroid, sclera, and optic nerve) were dissected
and homogenized, using a homogenizing probe for 5 minutes in 2 mL
ice-cold 20 mM Tris-HCl buffer (pH 7.5), per gram of tissue. Crude
homogenates for purification were prepared from whole bovine eyes using
a meat grinder followed by homogenization in a blender for 1 minute in
the previously mentioned buffer containing 2 mM EDTA, 100 mg/L
phenylmethylsulfonyl fluoride, 100 mg soybean trypsin inhibitor, and
200 mg/L benzamidine. These protease inhibitors were included in the
buffers up to the CaM-Sepharose-4B affinity chromatography step. The
homogenates were centrifuged at 10,000g for 25 minutes, and
the supernatant was filtered through glass wool.
2-Mercaptoethanol and EGTA were added to the supernatants of samples to
final concentrations of 10 and 0.1 mM, respectively.