Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle.
This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affinity chromatography and gel filtration in the presence of aprotinin. The final product (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70,000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at Mr 70,000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37,000 and 33,000 under reduced conditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic activity.
Matsuo
O
O
Department of Physiology, Kinki University School of Medicine, Osaka, Japan.
Fukao
H
H
Izaki
S
S
Matsuo
C
C
Ueshima
S
S
D002460:Cell Line; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D009215:Myometrium / Q000166:cytology / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000032:analysis; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000302:isolation & purification
D011960:Receptors, Estrogen; D010959:Tissue Plasminogen Activator