Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

Lodola, A.; Spragg, S. P.; Holbrook, J. John

doi:10.1042/bj1690577

Cited by 27 publications

(27 citation statements)

References 23 publications

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“…NADH and Malate Binding. We have confirmed previous reports that NADH fluorescence is enhanced and the protein's fluorescence is quenched when this ligand binds to sMDH (Holbrook & Wolfe, 1972;Lodola et al, 1978b). Additional fluorescence changes occur when malate is added to the enzyme-cofactor complex.…”

Section: Fmaxsupporting

confidence: 90%

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Cooperativity in the mechanism of malate dehydrogenase

Zimmerle

Alter²

1993

Biochemistry

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Cooperativity in the catalytic mechanism of porcine cytoplasmic malate dehydrogenase (sMDH) has been a point of ongoing discussion. Though previous investigations revealed little evidence of cooperativity, chemical modification studies reported by this laboratory demonstrate that binding of cofactor or cofactor plus substrate causes the enzyme's subunits to become chemically nonidentical. Therefore, we have reexamined the enzyme's steady-state kinetic and ligand-binding properties. To aide in characterizing sMDH kinetics, activities of the native enzyme and of sMDH, which was partially inactivated by an active-site-specific reagent, were examined. As expected for a negatively cooperative enzyme, steady-state kinetics (at pH 8.0, the pH optimum of the enzyme) are characterized by concave Eadie-Hofstee plots. Further, qualitative as well as quantitative results from partially inactivated sMDH strongly support negative cooperativity and eliminate many alternative mechanisms. Finally, results from equilibrium binding experiments are consistent with cytoplasmic malate dehydrogenase binding NADH in a negatively cooperative manner. Together, these results indicate that cytoplasmic malate dehydrogenase acts as a negatively cooperative enzyme.

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Section: Fmaxsupporting

confidence: 90%

“…Enzyme preparations had specific activities of 750-800 units (min-l) mg-l of protein when assayed in the direction of OAA reduction (0.25 mM NADH, 1 mM OAA, 50 mM HEPES, pH 8.0, and 25 "C). This compares favorably with other reported preparations (Johnson & Rupley, 1979;Lodola et al, 1978a;Glatthaar et al, 1974;Gerding & Wolfe, 1969).…”

supporting

confidence: 89%

Cooperativity in the mechanism of malate dehydrogenase

Zimmerle

Alter²

1993

Biochemistry

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“…Pig heart cytoplasmic MDH was prepared by method 2 of Lodola et al (1978) and was determined and assayed as described by those authors. Pig heart LDH was prepared as usual in the laboratory, and assayed as described by .…”

Section: Methodsmentioning

confidence: 99%

Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase

Lodola¹,

Parker²,

Jeck³

et al. 1978

Biochemical Journal

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1. The pH-dependencies of the binding of NADH and reduced nicotinamide--benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3--6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.

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“…For determination of stoichiometry, data at arrestin Ͼ Ͼ K D were analyzed by plotting 1/(1 Ϫ ) as a function of Rh*// Arr tot (40), where is the fractional saturation of arrestin, F/⌬F max .…”

Section: Methodsmentioning

confidence: 99%

“…The curve fits a 1:1 complex formed between arrestin-1 and monomeric P-Rh*. Linear transformation of the data (40), shown in the inset, also indicates a 1:1 stoichiometry of interaction. Thus, the binding of monomeric P-Rh* to arrestin-1 demonstrates high affinity and 1:1 stoichiometry characteristic of physiologically relevant interaction of arrestin-1 with P-Rh* in native disc membranes (26,50,51).…”

Section: Light-dependent Phosphorylation Of Monomeric Rhodopsinmentioning

confidence: 99%

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

Bayburt

Vishnivetskiy

McLean

et al. 2011

Journal of Biological Chemistry

174

177

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G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated lightactivated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.Visual phototransduction is quenched by a two-step mechanism. First, light-activated rhodopsin (Rh*) 3 is phosphorylated multiple times by GRK1. Arrestin-1 4 binding to active phosphorylated rhodopsin (P-Rh*) blocks further transducin activation (1) by steric exclusion (2). The binding of arrestin-1 to P-Rh* is an important molecular mechanism for signal shut-off (3). However, key details of the requirements for physical interaction of arrestin-1 with rhodopsin remain to be explored. Rhodopsin, which is highly concentrated in photoreceptor membranes, has been observed to form arrays of dimers, thus raising the possibility that the dimer is a functional unit (4). Although evidence of a preferred dimer interface has been reported (5), the functional role of rhodopsin oligomers remains controversial (6, 7). Accumulating evidence with other GPCRs indicates that oligomerization could be widespread (8 -10). Several models for rhodopsin dimers (11, 12) and monomers (6, 13) interacting with signaling partners transducin, rhodopsin kinase, and arrestin have been proposed. These models need rigorous experimental testing at different steps of the functional cycle of rhodopsin and other class A GPCRs (reviewed in Refs. 7 and 14). Modified high density lipoprotein particles (nanodiscs) consist of a phospholipid bilayer stabilized by a membrane scaffold protein (MSP) (15-17). These nanodiscs can be used to selectively isolate monomeric GPCRs imbedded into lipid bilayer. We (18) and others (19,20) have previously demonstrated that rhodopsin monomers incorporated in nanodiscs are highly functional in signaling to transducin. The same was shown for monomeric rhodopsin purified in detergent (dodecyl maltoside), where rhodopsin and transducin form a 1:1 complex (21). Howe...

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Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

Cited by 27 publications

References 23 publications

Cooperativity in the mechanism of malate dehydrogenase

Cooperativity in the mechanism of malate dehydrogenase

Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

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