An enzyme-coupled assay for glyoxylic acid

Carpenter, Sarah E.; Merkler, David J.

doi:10.1016/j.ab.2003.09.012

Cited by 7 publications

(7 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We subsequently developed a cell-free kinetic assay capable of measuring PAM amidating activity via spectrophotometric detection of converted glyoxylate, a by-product of the amidation reaction19,20. Matching each reaction for PAM input, we observed reduced amidating activity for p.Asp563Gly-PAM (p=1.0x10 -5 ) and p.Tyr651Phe-PAM (p=4.1x10 -6 ) (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells

et al. 2018

View full text Add to dashboard Cite

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human β-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in β-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Amidating activity was measured via spectrophotometric detection of chemically converted glyoxylate20. An assay mix of 100mM MES pH 5.5, 2μM CuSO 4 , 10mM L-ascorbic acid, 0.1mg/mL bovine liver catalase, and 20mM hippuric acid (Sigma-Aldrich) was split into glass vials.…”

Section: Methodsmentioning

confidence: 99%

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Malate: The enzyme-coupled colorimetric assay with malate dehydrogenase and phenazine methosulfate was used to shuttle electrons from the reduced NADH to thiazolyl blue tetrazolium bromide, detecting the formazan formed at 578 nm (modified from Carpenter & Merkler, 2003).…”

Section: Extraction and Determination Of Metabolitesmentioning

confidence: 99%

Effects of salts on aerobic metabolism ofDebaryomyces hansenii

Sánchez

Arreguín-Espinosa

Calahorra

et al. 2008

FEMS Yeast Research

View full text Add to dashboard Cite

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.

show abstract

“…We subsequently developed a cell-free kinetic assay capable of measuring PAM amidating activity via spectrophotometric detection of converted glyoxylate, a by-product of the amidation reaction (17,18). Matching each reaction for PAM input, we observed reduced amidating activity for D563G-PAM (p=1.0x10 -5 ) and Y651F-PAM (p=4.1x10 -6 ) ( Figure 1A).…”

Section: T2d-associated Pam Alleles Cause Pam Loss-of-functionmentioning

confidence: 99%

Type 2 Diabetes Risk Alleles Reveal a Role for Peptidylglycine Alpha-amidating Monooxygenase in Beta Cell Function

Raimondo

Thomsen

Hastoy

et al. 2017

Preprint

View full text Add to dashboard Cite

149Word count: 4500Number of tables and figures: 5 . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/158642 doi: bioRxiv preprint first posted online Jul. 3, 2017; 2 ABSTRACT Molecular mechanisms underpinning the genetic risk for type 2 diabetes (T2D) remain poorly understood, hindering translation into new therapies. Recently, genome-wide studies identified two coding variants in Peptidylglycine Alpha-amidating Monooxygenase (PAM) associated with T2D risk and measures of beta cell dysfunction. Here, we demonstrate that both risk alleles impact negatively on overall PAM activity, but via distinct effects on expression and catalytic function. In a human beta cell model, PAM silencing caused decreased insulin content and altered dynamics of granule exocytosis. Analysis of primary human beta cells from cadaveric donors confirmed an effect on exocytosis in carriers of the p.D563G T2D-risk allele. Finally, we show that the granular packaging protein Chromogranin A is a PAM substrate and a strong candidate for mediating downstream effects on insulin secretion. Taken together, our results establish a role for PAM in beta cell function, and uncover a novel mechanism for T2D-associated PAM alleles.

show abstract

An enzyme-coupled assay for glyoxylic acid

Cited by 7 publications

References 28 publications

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells

Effects of salts on aerobic metabolism ofDebaryomyces hansenii

Type 2 Diabetes Risk Alleles Reveal a Role for Peptidylglycine Alpha-amidating Monooxygenase in Beta Cell Function

Contact Info

Product

Resources

About