ID 61875
Author
Miki, Mizue University of Tokushima
Tanimoto, Kumi University of Tokushima
Shikata, Tomoko University of Tokushima
Keywords
Tissue plasminogen activator
cell culture
carbohydrate
animal cell
fibrinolysis
Content Type
Departmental Bulletin Paper
Description
To begin the examination of the role of human type carbohydrate moiety of tissue plasminogen activator
(t-PA) on the binding of the enzyme to fibrin,the naturally glycosylated enzyme was produced by microcarrier
culture of human cells established from normal uterine muscle. The cells grown on microcarrers in Haunks'
MEM supplemented with 10 % FBS (1.6 x 10 6 cells/ml) rapidly detached themselves from microcarrier in a
serum-free medium (t-PA production medium) within 5 days, and it was difficult to produce t-PA for long time
(t-PA production: an average of 3 IU/ml/day over 5 days). Addition of 0.5% beef extract to the serum-free
medium suppressed their detaching from microcarriers. By regulating the pH (7.4) and dissolved oxygen(4
ppm) of the serum free medium,the cell density of microcarrier culture increased to 1.2 x10 7 cells/ml and t-PA
was produced over 38 days (t-PA production: an average of 836 IU/ml/day over 38 days). Native t-PAs purified
to homogeneity from the culture broth had the molecular weight of 63,000 and 65,000 containing 5.6 and 8.5 %
carbohydrate,respectively (molecular mass of protein moiety was calculated to be 59,500). By enzymatic
digestion of carbohydrate moiety in native t-PA,we obtained partially deglycosylated t-PA with molecular
weights of 60,000 and 62,000. Completely deglycosylated t-PA was obtained by t-PA production in the
presence of 10μg/ml tunicamycin (N-glycosylation inhibitor). This suggests that N-glycosylationof t-PA
occurs while O-glycosylation is absent. The binding strength of these enzymes to fibrin increased with
decrease of the carbohydrate content. The carbohydrate moiety of human type glycosylated t-PA probably
modulates the binding strength of the enzyme to fibrin.
Journal Title
徳島大学総合科学部自然科学研究 = Natural Science Research, The University of Tokushima
ISSN
09146385
NCID
AN10065859
Publisher
徳島大学総合科学部
Volume
22
Start Page
97
End Page
104
Sort Key
97
Published Date
2008-12
EDB ID
FullText File
language
eng
departments
Bioscience and Bioindustry