Protein tyrosine phosphatase: enzymatic assays

Montalibet, Jacqueline; Skorey, Kathryn; Kennedy, Brian P.

doi:10.1016/j.ymeth.2004.07.002

Cited by 134 publications

(117 citation statements)

References 35 publications

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“…Such regulatory serine/threonine protein phosphatases are classified into four main enzymes; type 1 (PP1), type 2A (PP2A), type 2B (PP2B) and type 2C (PP2C) (Cohen 1989). Another phosphatase family is protein tyrosine phosphatase (PTPs), which removes phosphate groups from phosphorylated tyrosine residues of proteins (for a review, see Montalibet et al 2005). Both mouse and human spermatozoa contain highly active tyrosine phosphatases and inhibition of tyrosine phosphatases with sodium pervanadate, bis(N,N-dimethylhydroxoamido) hydroxovanadate, ethyl-3,4-dephostatin and phenylarsine oxide prevents the acrosome reaction, suggesting that PTPs play a role in mammalian sperm exocytosis (Tomes et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Ashizawa¹,

Wishart²,

Katayama³

et al. 2006

Reproduction

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The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 8C. In the presence of 2 mmol CaCl 2 /l at 40 8C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca 2C , motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 8C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca 2C . These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca 2C, was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca 2C and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca 2C plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.

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Section: Discussionmentioning

confidence: 99%

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Ashizawa¹,

Wishart²,

Katayama³

et al. 2006

Reproduction

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“…Protein tyrosine phosphatase activity was spectrometrically determined using p-nitrophenyl phosphate [11].…”

Section: Determination Of Protein Tyrosine Phosphatase Activitymentioning

confidence: 99%

Basal reactive oxygen species determine the susceptibility to apoptosis in cirrhotic hepatocytes

Raval

Lyman

Nitta

et al. 2006

Free Radical Biology and Medicine

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Hepatocytes from cirrhotic murine livers exhibit increased basal ROS activity and resistance to TGFβ-induced apoptosis, yet when ROS levels are decreased by antioxidant pretreatment, these cells recover susceptibility to apoptotic stimuli. To further study these redox events, hepatocytes from cirrhotic murine livers were pretreated with various antioxidants prior to TGFβ treatment and the ROS activity, apoptotic response, and mitochondrial ROS generation were assessed. In addition, normal hepatocytes were treated with low-dose H 2 O 2 and ROS and apoptotic responses determined. Treatment of cirrhotic hepatocytes with various antioxidants decreased basal ROS and rendered them susceptible to apoptosis. Examination of normal hepatocytes by confocal microscopy demonstrated co-localization of ROS activity and respiring mitochondria. Basal assessment of cirrhotic hepatocytes showed non-focal ROS activity that was abolished by antioxidants. After pretreatment with an adenovirus expressing MnSOD, basal cirrhotic hepatocyte ROS was decreased and TGFβ-induced co-localization of ROS and mitochondrial respiration was present. Treatment of normal hepatocytes with H 2 O 2 resulted in a sustained increase in ROS and resistance to TGFβ apoptosis that was reversed when these cells were pretreated with an antioxidant. In conclusion, cirrhotic hepatocytes have a non-focal distribution of ROS. However, normal and cirrhotic hepatocytes exhibit mitochondrial localization of ROS that is necessary for apoptosis.

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“…PTP1B activity was measured in rat and patient heart tissue with p‐nitrophenyl phosphate as substrate according to Montalibet et al 14. In brief, heart tissue (30–40 mg) was homogenated in lysis buffer (50 mmol/L of Bis‐Tris, 2 mmol/L of EDTA, 0.1 mmol/L of PMSF, 5 mmol/L of DTT, 0.5% Triton X‐100, and 20% glycerol), allowed to dissolve on ice, and protein content was determined.…”

Section: Methodsmentioning

confidence: 99%

Increased Protein Tyrosine Phosphatase 1B (PTP1B) Activity and Cardiac Insulin Resistance Precede Mitochondrial and Contractile Dysfunction in Pressure‐Overloaded Hearts

Nguyễn

Schwarzer

Schrepper

et al. 2018

JAHA

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BackgroundInsulin resistance in diabetes mellitus has been associated with mitochondrial dysfunction. Defects at the level of mitochondria are also characteristic of heart failure. We assessed changes in cardiac insulin response and mitochondrial function in a model of pressure overload‐induced heart failure.Methods and ResultsRats underwent aortic banding to induce pressure overload. At 10 weeks, rats showed cardiac hypertrophy and pulmonary congestion, but left ventricular dilatation and systolic dysfunction were only evident after 20 weeks. This contractile impairment was accompanied by mitochondrial dysfunction as shown by markedly reduced state 3 respiration of isolated mitochondria. Aortic banding did not affect systemic insulin response. However, insulin‐stimulated cardiac glucose uptake and glucose oxidation were significantly diminished at 10 and 20 weeks, which indicates cardiac insulin resistance starting before the onset of mitochondrial and contractile dysfunction. The impaired cardiac insulin action was related to a decrease in insulin‐stimulated phosphorylation of insulin receptor β. Consistently, we found elevated activity of protein tyrosine phosphatase 1B (PTP1B) at 10 and 20 weeks, which may blunt insulin action by dephosphorylating insulin receptor β. PTP1B activity was also significantly increased in left ventricular samples of patients with systolic dysfunction undergoing aortic valve replacement because of aortic stenosis.ConclusionsPressure overload causes cardiac insulin resistance that precedes and accompanies mitochondrial and systolic dysfunction. Activation of PTP1B in the heart is associated with heart failure in both rats and humans and may account for cardiac insulin resistance. PTP1B may be a potential target to modulate insulin sensitivity and contractile function in the failing heart.

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Protein tyrosine phosphatase: enzymatic assays

Cited by 134 publications

References 35 publications

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Basal reactive oxygen species determine the susceptibility to apoptosis in cirrhotic hepatocytes

Increased Protein Tyrosine Phosphatase 1B (PTP1B) Activity and Cardiac Insulin Resistance Precede Mitochondrial and Contractile Dysfunction in Pressure‐Overloaded Hearts

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