Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site.

Abstract

Calmodulin is a small, acidic, calcium-binding protein that exhibits multiple in vitro biochemical activities. Although calmodulin has no known enzymatic activity, it stimulates several enzyme activities in calcium-dependent manner. Because of its ubiquitous distribution and highly conserved structure, it has been difficult to elicit anti-calmodulin sera of useful titer. We describe here a reproducible and rapid method for producing anti-calmodulin sera. This method requires the injection of performic acid-oxidized calmodulin, but the antisera react equally well with unoxidized calmodulin. A response was elicited in 11 out of 11 rabbits using three variations of this method. Antisera titers were high enough to enable development of a quantitative radioimmunoassay using dilutions of whole sera, immunoglobulin fractions, or immunoglobulin fractions purified on calmodulin-Sepharose conjugates. For the majority of the antisera, the immunoreactive site is contained in a unique region of the calmodulin molecule. Based on the quantitative reactivity of overlapping tryptic and cyanogen bromide peptides, we propose that a major immunoreactive site is fund within an 18-residue region in the COOH-terminal domain of calmodulin.