Zahide DURAK, Metin GÜRÜ

Alkalen Fosfataz (Alp) Enziminin Özütlenmesi, İnhibisyonu Ve Kinetik Modellenmesi

Bu çalışma, sıçan karaciğerinden özütlenen alkalen fosfataz (ALP) enziminin kısmen özütlenmesini, inhibitörsüz ve inhibitör varlığında kinetik özelliklerinin araştırılmasını kapsamaktadır. Deneysel sonuçlar yardımıyla Michaelis Menten ve Lineweaver Burk grafikleri çizildi. Bu grafiklerden Km ve Vmax değerleri hesaplandı. Değerler: İnhibitörsüz Km= 0,047 Vmax=24,87; Üre inhibitörlü Km= 0,054 Vmax=20,83 ; Kreatinin inhibitörlü Km= 0,037 Vmax=20,83 olarak hesaplanmıştır. Bu değerlere göre enzimin substrata ilgisi ve maksimum hız tespit edildi. İnhibitör maddeyle deneyler tekrarlandı ve inhibisyon tipi belirlendi. Kandaki yüksek üre ve kreatinin seviyesinin alkalen fosfataz enzimini nasıl etkilediği bulundu. Kandaki ALP (Alkalen Fosfataz) üzerinde ürenininhibitör olarak kullanıldığı çalışmada reaksiyonun yarışmasız (nonkompetetif) bir reaksiyon, kreatininin inhibitör olarak kullanıldığı çalışmada ise reaksiyonun yarışmacı olmayan (unkompetetif) bir reaksiyonla gerçekleştiği tespit edilmiştir.

Anahtar Kelimeler:

Alkalen fosfataz, Michaelis Menten, yarışmasız inhibisyon, yarışmacı olmayan inhibisyon.

ISOLATION, INHIBITION AND KINETIC MODELLING OF ALKALINE PHOSPHATASE (ALP) ENZYME

This study scopes the purification of alkaline phosphatase enzyme extracted from the liver and the investigationof kinetic properties for the reactions with and without inhibitor. Michaelis Menten and Lineweaver Burk graphswere drawn by means of experimental results. Km and Vmax values were calculated from these graphics.According to these values, substrate affinity (Km) and maximum velocity (Vmax) values were determined. Valueswithout inhibitor; Km= 0,047 Vmax=24,87; with urea inhibitor Km= 0,054 Vmax=20,83 ; with creatinin inhibitorKm= 0,037 Vmax=20,83. The experiments were repeated with inhibitory substance and inhibition type wasestablished. How high blood urea and creatinine levels affected alkaline phosphatase activity were determined.In this study, urea was found to cause noncompetetive inhibition and creatinine to cause uncompetetiveinhibition on liver ALP.

Keywords:

Alkaline phosphatase Michaelis Menten, Lineweaver Burk, noncompetetive inhibition, uncompetetive inhibition,

PDF

___

Jakob A., Bryjak J., Wojtowicz H., Illeova V.,
Annus J., Polakovic M., “Inactivation kinetics
of food enzymes during ohmic heating”, Food
Chemistry, No 123, 369–376, 2010.
Biasutti M., Abuin E., Silber J., Correa M., Lissi
E., “Kinetics of reactions catalyzed by enzymes
in solutions of surfactants”, Advances in
Colloid and Interface Science, No 136, 1–24,
-
Wilin´ska A., Bryjak J., Illeova V., Polakovic
M., “Kinetics of thermal inactivation of alkaline
phosphatasein bovine and caprine milkand
buffer”, International Dairy Journal, No 17,
–586, 2007.
Ásgeirsson B., Guðjónsdóttir K., “Reversible
inactivation of alkaline phosphatase from
Atlantic cod (Gadus morhua) in urea”,
Biochimica et Biophysica Acta, No 1764, 190–
, 2006.
Van Hoof V., De Broe M.E., “Interpretation and
Clinical significance of alkaline phosphatase
isoenzyme patterns.” Critical Reviews in
Clinical Laboratory Sciences., 197-293, 1994.
B Behnke, M Kemper, H Kruse and D Muller-
Wiefel, “Bone alkaline phosphatase in children
with chronic renal failure”, Nephrology
Dialysis Transplantation, No 13, Oxford
University Press, Yayın 3, 662-667, 1998.
Becq F., Fanjul M., Merten M., “Possible
regulation of CFTR-chloride channels by
membrane-bound phosphates in pancreatic duct cells”, Proceeding of the National Academys
of Sciences of the United States of America
(PNAS), No 327, 337-342, 1993.
Day A. P., Feher M. D., Chopra R.,
“Triglyceride fatty acid chain lenght influences
the post prandial rise in serum intestinal alkaline
phosphatase activity”, Annals of Clinical
Biochemistry, No 29, 287-291, 1992.
Garrett RH, Grisham CM., Biochemistry,
Second Edition, Saunders College Publishing,
-427,1999.
Lehninger, A.L., Nelson, D.L., Cox, M.M.,
Lehninger principles of biochemistry, New
York: W.H. Freeman, 2005.
Leskovac, V., Comprehensive enzyme
kinetics, New York: Kluwer Academic/Plenum
Pub.,2003.
Walsh R, Martin E, Darvesh S., “Limitations of
conventional inhibitor classifications”, Integr
Biol (Camb)., No 3, 197-201,2011.
Stephenson FH., Calculations for Molecular
Biology and Biotechnology, Academic Pres,
An Imprint of Elsevier, Amsterdam, 2003.
Sutton R, Rockett B, Swindells P., Chemistry
for the Life Sciences, 1st ed., London, 2000.
Burtis CA, Ashwood ER, Bruns DE, Tietz
textbook of clinical chemistry and molecular
diagnosis, 4th ed. St. Louis: Elsevier Saunders,
-
Alan Wu., Tietz Clinical Guide to Laboratory
Tests, 4th ed. Elsevier Saunders, 2006.