Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca/sup 2 +/-calmodulin

Journal Article · Tue Aug 23 04:00:00 EDT 1988 · Biochemistry; (United States)

OSTI ID:6470140

LeVine, H III; Su, J L; Sahyoun, N E

A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca/sup 2 +/ for the interaction. Other divalent cations such as Zn/sup 2 +/, Mn/sup 2 +/, Cd/sup 2 +/, Co/sup 2 +/, and Ni/sup 2 +/ will substitute for Ca/sup 2 +/, while Mg/sup 2 +/ and Ba/sup 2 +/ will not. The antibody reacts with both the ..cap alpha..- and ..beta..-subunits on Western blots in a similar Ca/sup 2 +/-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of /sup 125/I Bolton-Hunter-labeled kinase with unlabeled enzyme. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the K/sub m app/ for calmodulin and an increase in the V/sub max/ for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the M/sub r/ 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca/sup 2 +/-dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca/sup 2 +/ and calmodulin act on the kinase molecule.

• Research Organization: Wellcome Research Labs., Research Triangle Park, NC (USA)
• OSTI ID: 6470140
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:17; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANTIBODIES
ATP
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CALCIUM COMPOUNDS
CATIONS
CENTRAL NERVOUS SYSTEM
CHARGED PARTICLES
CONFORMATIONAL CHANGES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
IONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MONOCLONAL ANTIBODIES
NERVOUS SYSTEM
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES