Enzymatic assay for calmodulins based on plant NAD kinase activity

Journal Article · Sun Jan 01 00:00:00 EST 1984 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(84)90441-X· OSTI ID:5887328

Harmon, A C; Jarrett, H W; Cormier, M J

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

• Research Organization: Univ. of Georgia, Athens
• OSTI ID: 5887328
• Journal Information: Anal. Biochem.; (United States), Vol. 140
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP-ASE
ENZYME ACTIVITY
PHOSPHODIESTERASES
PHOSPHOTRANSFERASES
PURIFICATION
PROTEINS
BIOASSAY
BRAIN
CALCIUM COMPOUNDS
CATTLE
COMPARATIVE EVALUATIONS
ERYTHROCYTES
EXPERIMENTAL DATA
HEXADECANOIC ACID
MAN
MUSHROOMS
PISUM
SENSITIVITY
TETRAHYMENA
ACID ANHYDRASES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
BACTERIA
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
CARBOXYLIC ACIDS
CENTRAL NERVOUS SYSTEM
CILIATA
DATA
DOMESTIC ANIMALS
ENZYMES
ESTERASES
FUNGI
HYDROLASES
INFORMATION
INVERTEBRATES
LEGUMINOSAE
MAMMALS
MATERIALS
MICROORGANISMS
MONOCARBOXYLIC ACIDS
NERVOUS SYSTEM
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS-GROUP TRANSFERASES
PLANTS
PRIMATES
PROTOZOA
RHIZOBIUM
RUMINANTS
TRANSFERASES
VERTEBRATES
550200* - Biochemistry