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Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork.

Junk AK, Goel M, Mundorf T, Rockwood EJ, Bhattacharya SK - Mol. Vis. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. ajunk@med.miami.edu

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 L-malate 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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Related in: MedlinePlus

Representative enzymatic activity analyses for aldolase, pyruvate kinase and isocitrate dehydrogenase. Activity measurements were performed on 10 TM tissue samples (5 µg total protein each) derived from normal (dotted bars) or glaucomatous donors (hollow bars). Colorimetric assay was performed for determination of enzymatic activities and standard deviation from 10 individual measurements have been presented. A: Activity assay for aldolase, B: Activity assay for pyruvate kinase, and C: Activity assay measurement for isocitrate dehydrogenase. D: Activity assay for malate dehydrogenase. Each bar represents the mean±standard deviation from ten independent experimental readings (ten samples in each group) and was found significantly different from 0.0 for each activity measurement by the one-sample t-test. The asterisk indicates a p<0.05.
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f1: Representative enzymatic activity analyses for aldolase, pyruvate kinase and isocitrate dehydrogenase. Activity measurements were performed on 10 TM tissue samples (5 µg total protein each) derived from normal (dotted bars) or glaucomatous donors (hollow bars). Colorimetric assay was performed for determination of enzymatic activities and standard deviation from 10 individual measurements have been presented. A: Activity assay for aldolase, B: Activity assay for pyruvate kinase, and C: Activity assay measurement for isocitrate dehydrogenase. D: Activity assay for malate dehydrogenase. Each bar represents the mean±standard deviation from ten independent experimental readings (ten samples in each group) and was found significantly different from 0.0 for each activity measurement by the one-sample t-test. The asterisk indicates a p<0.05.

Mentions: The activity measurements were performed using standard assay systems for each enzyme for 10 samples per group: normal control and primary open angle glaucoma. All normal TM tissue samples were derived from cadaver eyes from National Disease Research Interchange, Philadelphia, PA. The ophthalmic details for normal eyes followed the tissue delivery. Thus more (about 16 eyes) samples were collected and assayed but only those were included for which details showed definite evidence of ocular health and absence of disease. The trabecular meshwork aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively (Figure 1A-D). Decreased activity is consistent with inactivating modification or degradation of proteins in glaucoma. Microarray studies (GEO accession number GDS359) have been performed by several groups using glaucomatous and normal trabecular meshwork tissue. These microarray studies did not show any discernable difference in the expression level of aldolase and pyruvate kinase at the mRNA level. Our quantitative proteomic analysis also showed decreased levels of aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase which is consistent with activity measurement (Table 1). We did detect the peptides of the enzymes from excised protein bands that were also immunoreactive for 4-hydroxynonenal (HNE), however, we failed to detect the modified peptides. Our failure to detect the modified peptides may be due to the use of trypsin in the processing of tissues. The failure of detection is explicable based on the possibility that modification might involve lysine and arginine residues which will then not be digested by trypsin. Another possibility is relative low abundance of modified residues. The lipid peroxidation modified proteins have been shown to undergo rapid degradation [2]. Although western analysis will detect the modification due to amplification but it will be difficult to capture the modified peptides by mass spectrometry. Another complication that will also evade capture by mass spectrometry is heterogeneous nature of modification on proteins that will generate a large ensemble of different peptides each with a modification or the same peptide with a modification on different residues.


Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork.

Junk AK, Goel M, Mundorf T, Rockwood EJ, Bhattacharya SK - Mol. Vis. (2010)

Representative enzymatic activity analyses for aldolase, pyruvate kinase and isocitrate dehydrogenase. Activity measurements were performed on 10 TM tissue samples (5 µg total protein each) derived from normal (dotted bars) or glaucomatous donors (hollow bars). Colorimetric assay was performed for determination of enzymatic activities and standard deviation from 10 individual measurements have been presented. A: Activity assay for aldolase, B: Activity assay for pyruvate kinase, and C: Activity assay measurement for isocitrate dehydrogenase. D: Activity assay for malate dehydrogenase. Each bar represents the mean±standard deviation from ten independent experimental readings (ten samples in each group) and was found significantly different from 0.0 for each activity measurement by the one-sample t-test. The asterisk indicates a p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2904041&req=5

f1: Representative enzymatic activity analyses for aldolase, pyruvate kinase and isocitrate dehydrogenase. Activity measurements were performed on 10 TM tissue samples (5 µg total protein each) derived from normal (dotted bars) or glaucomatous donors (hollow bars). Colorimetric assay was performed for determination of enzymatic activities and standard deviation from 10 individual measurements have been presented. A: Activity assay for aldolase, B: Activity assay for pyruvate kinase, and C: Activity assay measurement for isocitrate dehydrogenase. D: Activity assay for malate dehydrogenase. Each bar represents the mean±standard deviation from ten independent experimental readings (ten samples in each group) and was found significantly different from 0.0 for each activity measurement by the one-sample t-test. The asterisk indicates a p<0.05.
Mentions: The activity measurements were performed using standard assay systems for each enzyme for 10 samples per group: normal control and primary open angle glaucoma. All normal TM tissue samples were derived from cadaver eyes from National Disease Research Interchange, Philadelphia, PA. The ophthalmic details for normal eyes followed the tissue delivery. Thus more (about 16 eyes) samples were collected and assayed but only those were included for which details showed definite evidence of ocular health and absence of disease. The trabecular meshwork aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively (Figure 1A-D). Decreased activity is consistent with inactivating modification or degradation of proteins in glaucoma. Microarray studies (GEO accession number GDS359) have been performed by several groups using glaucomatous and normal trabecular meshwork tissue. These microarray studies did not show any discernable difference in the expression level of aldolase and pyruvate kinase at the mRNA level. Our quantitative proteomic analysis also showed decreased levels of aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase which is consistent with activity measurement (Table 1). We did detect the peptides of the enzymes from excised protein bands that were also immunoreactive for 4-hydroxynonenal (HNE), however, we failed to detect the modified peptides. Our failure to detect the modified peptides may be due to the use of trypsin in the processing of tissues. The failure of detection is explicable based on the possibility that modification might involve lysine and arginine residues which will then not be digested by trypsin. Another possibility is relative low abundance of modified residues. The lipid peroxidation modified proteins have been shown to undergo rapid degradation [2]. Although western analysis will detect the modification due to amplification but it will be difficult to capture the modified peptides by mass spectrometry. Another complication that will also evade capture by mass spectrometry is heterogeneous nature of modification on proteins that will generate a large ensemble of different peptides each with a modification or the same peptide with a modification on different residues.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA. ajunk@med.miami.edu

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 L-malate 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.

Show MeSH
Related in: MedlinePlus