A RAPID, QUANTITATIVE ASSAY FOR MEASURING ALKALINE-PHOSPHATASE ACTIVITY IN OSTEOBLASTIC CELLS IN-VITRO

被引:186
作者
SABOKBAR, A [1 ]
MILLETT, PJ [1 ]
MYER, B [1 ]
RUSHTON, N [1 ]
机构
[1] UNIV CAMBRIDGE, ORTHOPAED RES UNIT, CAMBRIDGE, ENGLAND
来源
BONE AND MINERAL | 1994年 / 27卷 / 01期
关键词
ALKALINE PHOSPHATASE; ENZYME ASSAY; OSTEOBLAST; SAOS-2;
D O I
10.1016/S0169-6009(08)80187-0
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.
引用
收藏
页码:57 / 67
页数:11
相关论文
共 33 条
[1]  
BESSEY OA, 1946, J BIOL CHEM, V164, P321
[2]  
BKOJDT M, 1992, CYTOKINES BONE METAB, P1
[3]  
BONEWALD LF, 1992, J BIOL CHEM, V267, P8943
[4]  
BOYAN BD, 1989, J BIOL CHEM, V264, P11879
[5]   PRECIPITATION METHOD FOR SEPARATING AND QUANTIFYING BONE AND LIVER ALKALINE-PHOSPHATASE ISOENZYMES [J].
BURLINA, A ;
PLEBANI, M ;
SECCHIERO, S ;
ZANINOTTO, M ;
SCIACOVELLI, L .
CLINICAL BIOCHEMISTRY, 1991, 24 (05) :417-423
[6]   EFFECT OF HORMONES AND GROWTH-FACTORS ON ALKALINE-PHOSPHATASE ACTIVITY AND COLLAGEN-SYNTHESIS IN CULTURED RAT CALVARIAE [J].
CANALIS, E .
METABOLISM-CLINICAL AND EXPERIMENTAL, 1983, 32 (01) :14-20
[7]  
COLE DE, 1992, BONE, V1, P442
[8]  
CORNISHBOWDEN A, 1988, FOCUS SERIES
[9]  
DAVIES OL, 1987, STATISTICAL METHODS
[10]   SKELETAL ALKALINE-PHOSPHATASE SPECIFIC ACTIVITY IS AN INDEX OF THE OSTEOBLASTIC PHENOTYPE IN SUBPOPULATIONS OF THE HUMAN OSTEOSARCOMA CELL-LINE SAOS-2 [J].
FARLEY, JR ;
HALL, SL ;
HERRING, S ;
TARBAUX, NM ;
MATSUYAMA, T ;
WERGEDAL, JE .
METABOLISM-CLINICAL AND EXPERIMENTAL, 1991, 40 (07) :664-671