We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha 1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha h5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg(2+)-dependent and saturable. The relative order of affinity of nucleotide binding by G alpha h5 was GTP > GDP > ITP >> ATP > or = adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha h5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha h5 was recognized by a specific antibody to G alpha h7 or TGase II, indicating a homology with G alpha h (TGase II) family. These results demonstrate that 77-kDa G alpha h5 from pig heart is an alpha 1-adrenoceptor-coupled G alpha h (TGase II) family which has species-specificity in molecular mass.
Animal ; Binding Sites ; Binding, Competitive ; Cross Reactions ; GTP-Binding Proteins/metabolism* ; GTP-Binding Proteins/isolation & purification* ; GTP-Binding Proteins/immunology ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Myocardium/chemistry* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism ; Receptors, Adrenergic, alpha-1/metabolism ; Swine

OBJECTIVETo explore the difference of genes expression profiles between focal cerebral ischemia tissue and that treated with Baicalin using cDNA microarray.

METHODThe total RNAs were isolated from rat brains of sham-operation, vehicle (focal cerebral ischemia of rat brain) and baicalin-treated groups. mRNAs were reversely transcribed to cDNA with incorporation of fluorescent dUTP (Cy5 or Cy3 dUTP) to prepare hybridization probes. The PCR products of 4096 genes were spotted on the chip after a serial of treatment. The mixed probes were hybridized to the cDNA microarray. Axon Genepix 4000B and GenePixPro 3.0 software were used to scan and analyze the fluorescent signals.

RESULTThe expressions of 199 and 12 genes were found up-regulated and down-regulated, respectively, in the vehicle group compared with the sham-operation one. But the numbers of genes whose expressions were up-regulated and down-regulated were 89 and 88, respectively, when comparing the gene expression in the Baicalin-treated rat brain with that in the vehicle group. Moreover, one down-regulated and three up-regulated genes in the vehicle group were up-regulated and down-regulated in the Baicalin-treated group, respectively. Expressions of three up-regulated genes in the vehicle group were further reinforced in the Baicalin-treatment group.

CONCLUSIONMultiple pathways and nodes may be involved in the pharmacological effect of Baicalin on brain ischemia.

Animals ; Brain Ischemia ; genetics ; metabolism ; Flavonoids ; isolation & purification ; pharmacology ; GTP-Binding Proteins ; genetics ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation ; drug effects ; Male ; Oligonucleotide Array Sequence Analysis ; Plants, Medicinal ; chemistry ; Pyruvate Kinase ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Scutellaria ; chemistry ; Vimentin ; genetics ; metabolism