A restriction endonuclease assay for expression of human alpha-amylase isozymes.
BACKGROUND
The alpha-amylase isozymes can be detected separately by electrophoresis; however, sometimes the identification is difficult because of their microheterogeneity. In the present study, we tried to establish a convenient method for the detection of alpha-amylase isozyme expression.


METHODS
The procedure is based on three different restriction sites presented in those genes; a PstI site in both AMY 2A and 2B genes, a HaeII site in both AMY 1 and 2A genes, and a BamHI site in AMY 2B gene. After amplification from total tissue RNAs by RT-PCR with primers that were able to cover each exon, the products were cleaved with corresponding restriction endonucleases.


RESULTS
This method was applied to human samples from the parotid gland, liver (non-hepatoma), hepatoma and white blood cells (WBCs). The results indicated that the parotid gland and hepatoma (also liver) clearly expressed AMY 1 and AMY 2B genes, respectively. However, AMY 2B gene was also expressed apparently in WBCs, which produced salivary-type isozyme of the alpha-amylase, although the amylase protein was not able to identify for the hepatic isozyme.


CONCLUSIONS
The method presented here might be convenient and useful for the determination of alpha-amylase isozyme expression in humans.
Hokari
Shigeru
S
Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma, Saitama 350-0495, Japan. shhokar@saitama-med.ac.jp
Miura
Kae
K
Koyama
Iwao
I
Kobayashi
Minako
M
Komine
Shin-ichi
S
Komoda
Tsugikazu
T
D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics; D007962:Leukocytes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D010306:Parotid Gland / Q000201:enzymology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000516:alpha-Amylases / Q000032:analysis* / Q000235:genetics*
D007527:Isoenzymes; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D000516:alpha-Amylases