Phosphoinositide-specific Phospholipase C (PI-PLC) is a second messenger of signal transducer on cell membrane. In the previous study, PLC of bovine brain has been purified three isozymes. In this paper, uterus and seminal vesicle have been purified. Two peaks of PI-PLC activity were resolved when bovine uterus and seminal vesicle proteins were chromatographed on a DEAE and phenyl TSK 5PW HPLC column. Each two peak was compared with PI-PLC I, II and III from bovine brain and we got the retention time on HPLC. The peak fractions with PLC activity were tested homogeneity with brain PLC monoclonal antibodies (Mab). Mab-labeled affigels were bounded in the range of 73.8%~97.5% with PLC I, II and III. Homogeneity of fractions were revealed that DEAE F-1 and phenyl F-1-I were highest level of PLC III in uterus and seminal vesicle and DEAE F-2 and phenyl F-2-I were mixed PLC I and II.
Antibodies, Monoclonal ; Brain* ; Cell Membrane ; Chromatography, High Pressure Liquid ; Isoenzymes* ; Phospholipases* ; Second Messenger Systems ; Seminal Vesicle Secretory Proteins ; Seminal Vesicles* ; Transducers ; Type C Phospholipases* ; Uterus*

Although superoxide anions (O2.-) and H2O2 are generally considered to be toxic by-products of respiration, recent evidence suggests that the production of these reactive oxygen species (ROS) might be an integral component of membrane receptor signaling. In mammalian cells, a variety of extracellular stimuli have recently been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of stimulant-dependent signaling. In the next few years, therefore, a flurry of research activity is expected in relation to the elucidation of ROS production in response to receptor stimulation, identification of ROS target molecules, and investigation of ROS elimination. The goal of this report is to review our current knowledge of ROS-regulated signal transduction and propose future directions.
Animal ; Eukaryotic Cells/metabolism ; Human ; Hydrogen Peroxide/metabolism* ; Oxidation-Reduction ; Prokaryotic Cells/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction*

OBJECTIVES: Thioredoxin peroxidase(TPx), which does not exhibit similar activity and amino acid sequence homology to conventional antioxidant enzymes has been purified from S cerevisiae and bovine brain. Natural killer enhancing factor-A(NKEF-A)/ proliferation associated gene(PAG), natural killer enhancing factor-B(NKEF-B)/TPx and MER5 which has sequence homology to yeast TPx has been recently characterized biochemically, Prosperi has reported that the level of PAG in HL-60 cells was increased after serum stimulation and decreased after differentiation induced by DMSO treatment. It is well known that thioredoxin, the electron donor to thioredoxin peroxidase, also implicated in cell proliferation via protein kinase C pathway. Disturbed balance of reactive oxygen species and antioxidant in tumor tissue could enhance the cancer promotion This study was designed to investigate the distribution of NKEF-A/FAG, NKEF-B/TPx and MER5 in various tissues, and the expression of NKEF-A/PAG, NKEF-B/TPx and MER5 in human cancers. METHODS: We used antibodies against the purified recombinant protein of NKEF-A/PAG and NKEF-B/TPx and C-terminus amino acids(SP-TASKEYFEKVHO) of MER5, We separated cytosole and mitochondria from rat liver and prepared crude extract from these. We prepared crude extract of various tissues from rat and cancer tissue from lung, stomach and breast and paired normal tissue. Immunoblot analysis of these crude extracts was performed. RESULTS: 1) NKEF-A/PAG and NKEF-B/TPx existed in cytosolic fraction as Cu, Zn-SOD and MER5 mainly exist mainly in mitochondrial fraction as Mn-SOD. Although the level of NKEF-AIPAG, NKEF-B/TPx and MER5 was different, all tissues exhibited NKEF-A/PAG, NKEF-B/TPx and MER5 immunoreactive bands. The adrenal gland had relatively strong band of MEK. 2) The expression of NKEF-A/PAG in HL-60 cell was increased after serum stimulation and decreased after cell differentiation induced by DMSO treatment. 3) The expression of NKEF-A/PAG was increased in lung and breast cancer tissues compaired to paired normal tissues but was not changed in stomach cancer tissues and the expression of NKEF-B/TPx and MER5 was not changed in lung, stomach and breast cancer tissues compaired to paired normal tissues. 4) The level of NKEF-A/PAG, NKEF-B/TPx and MER5 was not different among various lung cancer cell lines. CONCLUSION: The NKEF-A/PAG, NKRF-B/TPx and MER5 are present in the cytosol and mitochondria of various tissues. The NKEF-A/PAG, in particular, is associated with cell proliferation and differentiation and overexpressed in lung and breast cancer.
Adrenal Glands ; Animals ; Antibodies ; Brain ; Breast ; Breast Neoplasms ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Complex Mixtures ; Cytosol ; Dimethyl Sulfoxide ; HL-60 Cells ; Humans* ; Liver ; Lung ; Lung Neoplasms ; Mitochondria ; Peroxidases* ; Peroxiredoxins ; Protein Kinase C ; Rats ; Reactive Oxygen Species ; Saccharomyces cerevisiae ; Sequence Homology ; Sequence Homology, Amino Acid ; Stomach ; Stomach Neoplasms ; Superoxide Dismutase ; Thioredoxins* ; Tissue Donors ; Yeasts