Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork

Anna Junk, Manik Goel, Tom Mundorf, Edward J. Rockwood, Sanjoy K Bhattacharya

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Purpose: To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls. Methods: Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61±8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63±4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of Lmalate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm. Results: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS-PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls. Conclusions: Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

Original languageEnglish
Pages (from-to)1286-1291
Number of pages6
JournalMolecular Vision
Volume16
StatePublished - Aug 20 2010

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Trabecular Meshwork
Isocitrate Dehydrogenase
Carbohydrate Metabolism
Fructose-Bisphosphate Aldolase
Pyruvate Kinase
NADP
Enzymes
Malate Dehydrogenase
hydrazine
Oxaloacetic Acid
Hydrazones
Proteins
Trabeculectomy
Citric Acid Cycle
Open Angle Glaucoma
Coenzymes
Pyruvic Acid
L-Lactate Dehydrogenase
Cadaver
Glaucoma

ASJC Scopus subject areas

  • Ophthalmology

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Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork. / Junk, Anna; Goel, Manik; Mundorf, Tom; Rockwood, Edward J.; Bhattacharya, Sanjoy K.

In: Molecular Vision, Vol. 16, 20.08.2010, p. 1286-1291.

Research output: Contribution to journalArticle

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abstract = "Purpose: To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls. Methods: Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61±8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63±4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1{\%} genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of Lmalate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm. Results: Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS-PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls. Conclusions: Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.",
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