Assessment of protein kinase C isozymes by enzyme immunoassay and overexpression of type II in thyroid adenocarcinoma.
A two site enzyme immunoassay which quantitatively identifies types I, II, and III of protein kinase C isozymes has been designed. The soluble protein kinase C isozymes were selectively immobilized by type-specific monoclonal antibodies, MC-1a, -2a, and -3a (H. Hidaka et al., J Biol. Chem., 263: 4523-4526, 1988) which bind to the regulatory domain (NH2-terminal side) of protein kinase C. The amount of each isozyme was then determined using a horseradish peroxidase-conjugated polyclonal antibody raised against the COOH-terminal peptide of protein kinase C. By adding increasing concentrations of the antigen, the range of the assay proved to be 0.51-51, 0.081-8.1, and 0.31-31 nM for types I, II, and III, respectively. This sandwich method was used to determine the level of protein kinase C isozymes in rabbit tissues. Type I was mainly present in the cerebrum and cerebellum; the highest amount of type II isozyme was present in blood platelets [26.0 +/- 3.8 (SE) micrograms/g wet tissue]. We compared the protein kinase C isozyme levels in human normal thyroid gland and thyroid cancer tissues and found that type II protein kinase C specifically increased in thyroid cancer tissues. Immunocytochemical examination using MC-2a revealed that the cytoplasm of the cancer cells showed prominent immunoreactivity for type II isozyme.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
M
M
Hagiwara
T
T
Hachiya
M
M
Watanabe
N
N
Usuda
F
F
Iida
K
K
Tamai
H
H
Hidaka
D000230:Adenocarcinoma / Q000201:enzymology* / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000032:analysis* / Q000096:biosynthesis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D010455:Peptides / Q000138:chemical synthesis; D011493:Protein Kinase C / Q000032:analysis* / Q000096:biosynthesis; D011817:Rabbits; D013964:Thyroid Neoplasms / Q000201:enzymology* / Q000473:pathology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D010455:Peptides; D011493:Protein Kinase C