Determination of rat liver microsomal glucose-6-phosphatase activity: Study of citrate and G-6-P inhibition

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...

Section: Methodsmentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

“…Ca 2ϩ -activated ATP hydrolysis was measured by following the liberation of inorganic phosphate by the Baginski method (33,42). The various CaCl 2 concentrations used to obtain the required free Ca 2ϩ concentration were calculated with the MAXC computer software using the dissociation constants therein (43).…”

Section: Methodsmentioning

confidence: 99%

Dissection of the Functional Differences between Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) 1 and 3 Isoforms by Steady-state and Transient Kinetic Analyses

Dode

Vilsen

Baelen

et al. 2002

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca 2؉ -transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca 2؉ in activation of the overall reaction due to a decreased E 2 to E 1 Ca 2 transition rate and an increased rate of Ca 2؉ dissociation from E 1 Ca 2 . At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca 2؉ level (either an enhanced efflux of lumenal Ca 2؉ through the pump in E 2 form or insensitivity to inhibition by lumenal Ca 2؉ ). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca 2؉ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E 2 P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit membrane-spanning P-type ATPases that mediate the uphill transport of cytoplasmic Ca 2ϩ into the lumen of intracellular stores with a stoichiometry of two Ca 2ϩ per ATP hydrolyzed (1-3). Studies of the SERCA1a enzyme have shown that Ca 2ϩ transport and ATP utilization are coupled through long-range intramolecular interactions between the 10-helix transmembrane domain containing the Ca 2ϩ binding sites and the cytoplasmic actuator, phosphorylation, and nucleotidebinding domains (1-4). In the reversible catalytic cycle (5), the binding of the two calcium ions with high affinity from the cytoplasmic side of the membrane triggers the phosphorylation of Asp 351 by ATP, whereas the dephosphorylation occurs when the translocated calcium ions have been released from lowaffinity lumenally facing sites in exchange with protons being countertransported (Scheme 1).The SERCAs have long been known as key enzymes in the cytoplasmic Ca 2ϩ signaling events, and more recent studies have also drawn attention to the importance of the luminal Ca 2ϩ content for Ca 2ϩ signaling, folding and processing of newly synthesized proteins, and control of cell growth and apoptosis (6 -9). In addition to the functionally well characterized SERCA1a isoform, the 3 human SERCA genes encode at least 8 other SERCA proteins by alternative splicing (10 -15). Whereas SERCA2b seems to be a ubiquitously expressed housekeeping enzyme, the other isoforms are limited to specific c...

“…The ATP hydrolysis catalyzed by the microsomes was determined by measuring the amount of P i liberated (12) using the method of Baginski et al (19). ATP hydrolysis referable to Ca 2ϩ -ATPase activity was calculated following subtraction of the amount of P i liberated in the presence of 4 mM EGTA without added Ca 2ϩ .…”

Section: Methodsmentioning

confidence: 99%

Importance of Stalk Segment S5 for Intramolecular Communication in the Sarcoplasmic Reticulum Ca2+-ATPase

Sørensen

Andersen

2000