Organ-specific regulation of phosphofructokinase during facultative anaerobiosis in the marine whelk Busycotypus canaliculatum

1991 ◽  
Vol 69 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Ross E. Whitwam ◽  
Kenneth B. Storey
Keyword(s):  
Protein Kinase ◽  
Active Form ◽  
Covalent Modification ◽  
Retractor Muscle ◽  
Affinity Constant ◽  
Enzyme Analysis ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Fructose 2

The kinetic properties of 6-phosphofructo-1-kinase (PFK) were assessed in five organs (ventricle, radular retractor muscle, gill, hepatopancreas, and kidney) of aerobic and anoxic (21 h in N2-bubbled seawater) whelks, Busycotypus canaliculatum. The enzyme in all organs showed a stable modification of kinetic parameters as a result of exposure of the animal to anoxic conditions. In most cases these changes were consistent with the conversion of the enzyme to a less active form in the anoxic organ. In ventricle, for example, the anoxic enzyme form showed significant changes, including a 36% increase in the value of the substrate affinity constant (S0.5) for Mg∙ATP, a 19% increase in S0.5 fructose-6-phosphate, a 57% increase in the 50% inhibition value (I50) for phosphoenolpyruvate, a 30% increase in I50 citrate, and a fivefold increase in the activator constant (Ka) for fructose-2, 6-bisphosphate, as compared with the aerobic enzyme. Analysis of the time course of anoxia-induced modification of PFK showed that changes to the properties of gill PFK were accomplished within 2 h of the exposure to N2-bubbled seawater, whereas changes to ventricle PFK required up to 8 h. In vitro incubation of ventricle homogenates with Mg∙ATP plus protein kinase second messengers or with Mg2+ plus added protein phosphatases showed that the aerobic enzyme form was modified by protein kinase action with an increase in Ka fructose-2,6-bisphosphate that mimicked the effect of the aerobic to anoxic transition on the enzyme. Phosphatase action on the anoxic enzyme form had the opposite effect. The data suggest that the modification of PFK properties under anoxia in whelk organs is due to protein phosphorylation of the enzyme. Such covalent modification of PFK and other enzymes, notably pyruvate kinase, coordinates the anoxia-induced glycolytic rate depression and overall metabolic arrest that is a prominent feature of facultative anaerobiosis in marine molluscs.

Regulation of Fructose 1,6-Bisphosphatase Activity of Chlorella by Mole Mass Change

1996 ◽  
Vol 51 (9-10) ◽  
pp. 639-645 ◽  
Author(s):  
N. Grotjohann
Keyword(s):  
Enzyme Regulation ◽  
Fold Increase ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Phosphorylated Compounds ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma ◽  
Fructose 1,6 Bisphosphate

Fast protein liquid chromatography on Superose 6 of partially purified FBPase II from Chlorella reveals a 1350 kDa-form at pH 6.0 and a 67 kDa-form at pH 8.5. Treatment of the large enzyme form with 5mᴍ concentrations of Mg2+, F1,6P2, DTT or ATP leads to dissociation into smaller ones of 215 -470 kDa. Aggregation/dissoziation is a reversible process, as has been shown for the effect of F1,6P2 and of pH, by rechromatography. The change in mole mass results in alterations of the activitiy and of the kinetic properties of the enzyme forms, obtained. Dissociation results in a 4 - 6 fold increase in activity, as can be shown for F1,6P2-treated samples. Halfsaturation constants, as well as the degree of cooperativity of the 67- and the 1350- kDa form, are different for substrate affinity, activation by Mg2+ and DTT, and for inhibition by ATP. Both enzyme forms hydrolyse fructose 1,6 bisphosphate and seduheptulose 1,7 bisphosphate better than other phosphorylated compounds. The ratio of F1,6P2- to SDP-cleavage is 100:58 for the small enzyme form and 100: 84 for the large one. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme, generated by changes in the concentration of intermediates and effectors in the chloroplast stroma, leading to dissociation or aggregation. The conclusion is drawn that oligomerization of key enzymes, resulting in enzyme forms with different activities and different kinetic properties, might provide an effective mechanism for enzyme regulation in vivo

scholarly journals Pyruvate Kinase from the Land Snail Otala Lactea: Regulation by Reversible Phosphorylation During Estivation and Anoxia

1990 ◽  
Vol 154 (1) ◽  
pp. 321-337 ◽  
Author(s):  
ROSS E. WHITWAM ◽  
KENNETH B. STOREY
Keyword(s):  
Protein Kinase ◽  
Pyruvate Kinase ◽  
Time Course ◽  
Land Snail ◽  
Active Enzyme ◽  
Kinetic Properties ◽  
Reversible Phosphorylation ◽  
Enzyme Form ◽  
Otala Lactea ◽  
Activity State

Pyruvate kinase (PK) from tissues of the desert snail Otala lactea (Müller) undergoes a stable modification of its kinetic properties during estivation or in response to anoxia stress. In foot muscle and mantle, the kinetic changes induced by either state were virtually identical and were consistent with a less active enzyme form in estivation or anoxia: S0.5 PEP increased, and I50 values for Mg-ATP and L-alanine decreased, compared to the enzyme in control (aroused) snails. Estivation and anoxia also changed the properties of PK from hepatopancreas; some changes were consistent with a more active enzyme form (So.5 PEP decreased, I50 values for Mg-ATP and L-alanine increased) but the enzyme lost all sensitivity to the potent activator fructose-l,6-bisphosphate. A time course of changes in I50 Mg-ATP for foot PK and S0.5 PEP for hepatopancreas PK revealed that estivation-induced changes in enzyme properties occurred between 12 and 48 h after snails were deprived of access to food and water, whereas the reversal of these changes occurred within as little as lOmin in foot muscle after arousal was initiated. The molecular basis of the stable modification of PK kinetics appears to be reversible protein phoshorylation. The action of added cyclic-AMP-dependent protein kinase on foot or hepatopancreas PK from control (aroused) snails changed PK kinetic parameters to those characteristic of the enzyme form in estivating animals; the addition of stimulators of endogenous cyclic-GMPdependent protein kinase or protein kinase C had the same effect. Conversely, treatment with added phosphatases reconverted the properties of foot muscle PK from estivating snails to those characteristic of the control enzyme. The data suggest that reversible phosphorylation control over the activity state of regulatory enzymes of glycolysis is one mechanism contributing to the overall metabolic rate depression of the estivating state.

scholarly journals Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades

2004 ◽  
Vol 164 (3) ◽  
pp. 353-359 ◽  
Author(s):  
Nick I. Markevich ◽  
Jan B. Hoek ◽  
Boris N. Kholodenko
Keyword(s):  
Protein Kinase ◽  
Feedback Regulation ◽  
Kinetic Mechanism ◽  
Covalent Modification ◽  
Mitogen Activated Protein Kinase ◽  
Kinetic Properties ◽  
Stable States ◽  
Protein Activity ◽  
Bistable Switch ◽  
Mapk Cascades

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal–regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux

2019 ◽  
Vol 166 (4) ◽  
pp. 309-315 ◽  
Author(s):  
Taro Watanabe ◽  
Noriyuki Kioka ◽  
Kazumitsu Ueda ◽  
Michinori Matsuo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Translational Regulation ◽  
Cholesterol Efflux ◽  
Degradation Rate ◽  
Active Form ◽  
Density Lipoprotein ◽  
Protein Levels ◽  
Kinase C ◽  
Pkc Activation

Abstract ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

Active Form of the Protein Kinase CK2 α2β2 Holoenzyme Is a Strong Complex with Symmetric Architecture

2013 ◽  
Vol 9 (2) ◽  
pp. 366-371 ◽  
Author(s):  
Graziano Lolli ◽  
Alessandro Ranchio ◽  
Roberto Battistutta
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Active Form ◽  
Kinase Ck2

scholarly journals Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation

1995 ◽  
Vol 309 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J L Rosa ◽  
J X Pérez ◽  
F Ventura ◽  
A Tauler ◽  
J Gil ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Covalent Modification ◽  
Terminal Region ◽  
Bifunctional Enzyme ◽  
Dependent Protein Kinase ◽  
Adp Ribosylation ◽  
Camp Dependent Protein Kinase ◽  
Arginine Residues ◽  
Dependent Protein

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.

Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

1999 ◽  
Vol 202 (12) ◽  
pp. 1667-1675
Author(s):  
B. Michaelidis ◽  
A. Pallidou ◽  
P. Vakouftsi
Keyword(s):  
Pyruvate Kinase ◽  
Active Form ◽  
Land Snail ◽  
Land Snails ◽  
Kinetic Properties ◽  
Acid Base ◽  
Down Regulation ◽  
Helix Lucorum ◽  
End Products ◽  
Acid Base Status

The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

scholarly journals Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation

1993 ◽  
Vol 292 (2) ◽  
pp. 379-384 ◽  
Author(s):  
N Ben-Arie ◽  
M Khen ◽  
D Lancet
Keyword(s):  
Phase Ii ◽  
Olfactory Epithelium ◽  
Covalent Modification ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Lipid Solubility ◽  
Glutathione S Transferase ◽  
Pcr Cloning ◽  
Biotransformation Enzyme ◽  
Aldehydes And Ketones

The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process.

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