6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes

Şükrü BEYDEMİR

Makale detayları

6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes

Yazarlar:

Şükrü BEYDEMİR

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Veterinary and Animal Sciences 2004 Vol.28 Issue 4, pp.707-714

Belge Tipi:

Makale

Yayın Tarihi:

3/2004

Belge Dili:

Türkçe

Document ID (Org No.):

2000769460

Sıklık:

İki Aylık

eISSN:

1303-6181

pISSN:

1300-0128

Anahtar kelimeler:

Özet:

 

In this paper, a simple and rapid method for the purification of 6-phosphogluconate dehydrogenase from rat erythrocytes together with an analysis of the kinetic behavior and some properties of the enzyme are considered. The purification steps comprised high-speed centrifugation, 20-50% ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity gel chromatography. The yield was 78.4% and the specific enzyme activity was 5.15 EU/mg proteins. The molecular mass of the subunit was estimated to be 59,566 Da by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and native enzyme was found to be 111,000 Da by gel filtration column chromatography. The enzyme had an optimal pH at 7.0 and stable pH at 8.0 in 1 M Tris-HCl buffer, and optimal temperature at 45 ºC. KM and Vmax for NADP+ and 6-PGA as substrates were also determined. The inhibitor effects of ATP, NADPH and NADH were also examined, and Ki values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.

 

In this paper, a simple and rapid method for the purification of 6-phosphogluconate dehydrogenase from rat erythrocytes together with an analysis of the kinetic behavior and some properties of the enzyme are considered. The purification steps comprised high-speed centrifugation, 20-50% ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity gel chromatography. The yield was 78.4% and the specific enzyme activity was 5.15 EU/mg proteins. The molecular mass of the subunit was estimated to be 59,566 Da by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and native enzyme was found to be 111,000 Da by gel filtration column chromatography. The enzyme had an optimal pH at 7.0 and stable pH at 8.0 in 1 M Tris-HCl buffer, and optimal temperature at 45 ºC. KM and Vmax for NADP+ and 6-PGA as substrates were also determined. The inhibitor effects of ATP, NADPH and NADH were also examined, and Ki values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.

Makale detayları

6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes

Yazarlar:

Şükrü BEYDEMİR

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Veterinary and Animal Sciences 2004 Vol.28 Issue 4, pp.707-714

Belge Tipi:

Makale

Yayın Tarihi:

3/2004

Belge Dili:

Türkçe

Document ID (Org No.):

2000769460

Sıklık:

İki Aylık

eISSN:

1303-6181

pISSN:

1300-0128

6PGD

rat

erythrocyte

purification

kinetic properties

Sayı

Makale detayları

6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes

Yazarlar: Şükrü BEYDEMİR

Yayıncı: TUBITAK

Dergi: Turkish Journal of Veterinary and Animal Sciences 2004 Vol.28 Issue 4, pp.707-714

Belge Tipi: Makale

Yayın Tarihi: 3/2004

Belge Dili: Türkçe

Document ID (Org No.): 2000769460

Sıklık: İki Aylık

eISSN: 1303-6181

pISSN: 1300-0128

6PGD

rat

erythrocyte

purification

kinetic properties

Sayı

Yazarlar:

Şükrü BEYDEMİR

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Veterinary and Animal Sciences 2004 Vol.28 Issue 4, pp.707-714

Belge Tipi:

Makale

Yayın Tarihi:

3/2004

Belge Dili:

Türkçe

Document ID (Org No.):

2000769460

Sıklık:

İki Aylık

eISSN:

1303-6181

pISSN:

1300-0128