Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats.
Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.
Department of Biochemistry, Medical University of South Carolina, Charleston 29425.
J
J
Chao
K X
KX
Chai
L M
LM
Chen
W
W
Xiong
S
S
Chao
C
C
Woodley-Miller
L X
LX
Wang
H S
HS
Lu
L
L
Chao
D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006973:Hypertension / Q000378:metabolism*; D007249:Inflammation; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D011971:Receptors, Immunologic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012016:Reference Values; D012689:Sequence Homology, Nucleic Acid; D015843:Serpins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D002352:Carrier Proteins; D011971:Receptors, Immunologic; D015843:Serpins; C051324:kallistatin; D004247:DNA; D007610:Kallikreins