Pyruvate carboxylase: affinity labelling of the pyruvate binding site.

The active-site-directed reagent, bromopyruvate has been used to covalently label the pyruvate binding site of pyruvate carboxylase (E.C.6.4.1.1.) isolated from sheep liver. Oxalo-acetate proved to be the most effective reaction component in protecting the enzyme against inactivation; pyruvate was less effective although its efficiency was enhanced by the presence of acetyl CoA. The other reaction components, MgATP^2? and HCO₃^? failed to protect the enzyme against inactivation. Using bromo[2¹⁴C]pyruvate, it was shown that at 100% inactivation, 1.5 pyruvyl residues were bound per mole of biotin and when the reaction was carried out in the presence of acetyl CoA, this ratio was reduced to 1.0. Analysis of pronase digests of the enzyme revealed that more than 90% of the radioactivity was present as carboxy-hydroxyethyl cysteine.     

Specific affinity labelling of tubulin with bromocolchicine

Bromocolchicine, synthesized by substituting tho N-acetyl moiety of colchicine with a reactive bromoacetyl group, was found to be an affinity label for tubulin. Binding of [³H]colchicine to tubulin was competitively and irreversibly inhibited by bromocolchicine with a K_i value of 2.3 × 10^?5m. The affinity label could not be displaced by precipitating the protein with trichloroacetic acid and is thus covalently bound. Autoradiographs of brain high-speed supernatant proteins after their electrophoretic separation on sodium dodecyl sulphate/polyacrylamide gels showed that [³H]bromocolchicine reacted with four proteins, of which tubulin was one.Labelling of two of these proteins could be prevented by pretreatment of the brain extracts with α-bromoacetic acid, after which 70% of the covalently bound label was specifically located in the tubulin band. Up to 1.6 mol of affinity label could be bound per mol of tubulin, while under our experimental conditions 1 mol of protein bound irreversibly only 0.2 mol of [³H]colchicine. Autoradiography of sodium dodecyl sulphate/urea-polyacrylamide gels, which separate the subunits of tubulin, showed about 30% [³H] bromocolchicine bound to the α-subunit of tubulin and 70% to tho β-subunit.The irreversible binding site of colchicine was localized to the α-subunit, as labelling of only this subunit was inhibited by colchicine at high affinity label concentrations. At lower concentrations, colchicine inhibited the labelling of both subunits.Bromoacetic acid did not inhibit the reaction of the affinity label with the tubulin subunits, but increased the inhibition of [³H]bromocolchicine binding at lower concentrations of the affinity label in brain extracts preincubated with cold colchicine. This is interpreted to show a conformational change which takes place in the two subunits of tubulin upon binding of colchicine and results in the exposure of some of the binding sites of [³H]bromocolchicine to bromoacetic acid.     

An imidoester spin probe of membrane protein interactions: application to cytochrome c

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10^?9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10^?9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10^?9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.     

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB₂ by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB₂ in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB₂.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

Affinity labeling of specific regions of 23 S RNA by reaction of N-bromoacetyl-phenylalanyl-transfer RNA with Escherichia coli ribosomes

Reaction of the affinity-labeling reagent N-bromoacetyl-[¹⁴C]phenylalanyl-tRNA with Escherichia coli ribosomes results in covalent labeling of 23 S ribosomal RNA in addition to the previously reported labeling of ribosomal proteins. The reaction with the 23 S RNA is absolutely dependent on the presence of messenger RNA. Covalent attachment of the affinity label to 23 S RNA was demonstrated by its integrity in strongly dissociating solvents, and the conversion of the labeled material to small oligonucleotides by ribonuclease treatment. After digestion of labeled 23 S RNA with T₁ ribonuclease, the radioactivity is found mainly in two oligonucleotide fragments. These results support models in which both ribosomal RNA and ribosomal protein contribute to the structure of the region of the ribosome surrounding the peptidyl transferase center.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane

³H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3–4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-P_i' exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000–40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.Abbreviations NPA 2-azido-4-nitrophenol - DNP 2,4-dinitrophenol - DCCD N, N¹-dicyclohexylcarbodiimide - AE particles=bovine heart submitochondrial particles prepared by treatment with NH₄OH and EDTA at pH 8.8 - RCI respiratory control index - BSA bovine serum albumin     

Incorporation of specifically labeled cysteine into Escherichia coli protein

Protein obtained from several strains of Escherichia coli grown in the presence of [3,3′-¹⁴C]cystine contained the radiolabel in nearly all the other amino acids, suggesting catabolism of cysteine to pyruvic acid. Utilization in amino acid synthesis of the pyruvate thus generated can be blocked by growing the bacteria in a medium specifically enriched with most of the naturally occurring amino acids. Cysteine that is incorporated intact is diluted by de novo synthesis at low cystine concentrations; also, it was found that E. coli can use the sulfur of methionine for cysteine biosynthesis. Both of these latter two processes can be prevented by supplying an excess of exogenous cystine. This regiment leads to protein that is highly specifically labeled in the cysteine residues, with a minor amount (20–25%) of the label also appearing in alanine residues. Although this strategy was developed expressly for cysteine, it may be useful for incorporating other labeled amino acids that are also readily catabolized.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

Stable isotope labeling method targeting terminal tyrosine for relative peptide quantitation using mass spectrometry

Many neuropeptides lack suitable amino acid residues for modification by existing selective isotope labeling methods and use in relative quantitation by mass spectrometry. To address this issue, a new stable isotope labeling method that targets tyrosine residues by coupling with light cysteine (d₀) or heavy cysteine (d₂) in the presence of tyrosinase was developed. Optimal derivatization conditions for 1 μM leucine-enkephalin were achieved when 10 mM cysteine and 200 U/ml tyrosinase at pH 6.8 to 7.2 were used for a 60-min incubation period at room temperature. Under these conditions, leucine-enkephalin present at concentrations as low as 125 nM was successfully labeled. When comparisons between the lightly labeled (d₀) and heavily labeled (d₂) forms were made, a discrepancy between the actual concentration ratio and the raw peak intensity ratio was observed; this is due to the overlap of an isotopic peak of the d₀ with the monoisotopic peak of d₂. Fortunately, this discrepancy can be corrected by one of two simple computational approaches described. The quantitative labeling of this method to neuropeptides with the terminal tyrosine was confirmed and provides an alternative when other selective isotope-coded affinity tagging methods are not suitable.     

Labeling of the active site of cytoplasmic aspartate aminotransferase by -chloro-L-alanine

Syncatalytic inactivation of pig heart cytoplasmic aspartate aminotransferase by β-chloro-[U-¹⁴C]L-alanine resulted in the incorporation of radioactivity corresponding to one mole of the label per mole of the monomeric unit of the enzyme. A borohydride-reduced and then carboxymethylated preparation of the labeled enzyme was digested by trypsin. A radioactive peptide was isolated and found to contain a covalently linked pyridoxyl derivative which absorbed at 325 nm. The amino acid sequence of this peptide was Tyr-Phe-Val-Ser-Glu-Gly-Phe -Glu-Leu-Phe-Cys-Ala-Gln-Ser-Phe-Ser-Lys-Asn-Phe-Gly-Leu-Tyr-Asn-Glu-Arg. In the peptide the phosphopyridoxyl group seems to be covalently bound via alanyl moiety derived from β-chloro-L-alanine, the β-carbon atom of which is covalently linked to the ?-nitrogen atom of the lysyl residue(Lys). From a comparison with the amino acid composition of the phosphopyridoxyl peptide isolated from the tryptic digest of a borohydride-reduced holoenzyme, it was concluded that the modified lysul residue was identical to that involved in binding pyridoxal phosphate to the apoenzyme.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.     

THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE

An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 10⁶ iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10–15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or ¹²⁵I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 10⁶ iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with ¹²⁵I and ¹³¹I does not reveal the appearance of previously unexposed proteins.     

The Phosphorylation of Ribosomal Protein in Lemna minor

Sterile cultures of Lemna minor have been labeled with ³²P₁, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with ³²P₁, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome.     

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H₄PteGlu₅) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H₄PteGluB and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H₄PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.     

Can dimedone be used to study selenoproteins? An investigation into the reactivity of dimedone toward oxidized forms of selenocysteine

Dimedone is a widely used reagent to assess the redox state of cysteine‐containing proteins as it will alkylate sulfenic acid residues, but not sulfinic acid residues. While it has been reported that dimedone can label selenenic acid residues in selenoproteins, we investigated the stability, and reversibility of this label in a model peptide system. We also wondered whether dimedone could be used to detect seleninic acid residues. We used benzenesulfinic acid, benzeneseleninic acid, and model selenocysteine‐containing peptides to investigate possible reactions with dimedone. These peptides were incubated with H₂O₂ in the presence of dimedone and then the reactions were followed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS). The native peptide, H‐PTVTGCUG‐OH (corresponding to the native amino acid sequence of the C‐terminus of mammalian thioredoxin reductase), could not be alkylated by dimedone, but could be carboxymethylated with iodoacetic acid. However the “mutant peptide,” H‐PTVTGAUG‐OH, could be labeled with dimedone at low concentrations of H₂O₂, but the reaction was reversible by addition of thiol. Due to the reversible nature of this alkylation, we conclude that dimedone is not a good reagent for detecting selenenic acids in selenoproteins. At high concentrations of H₂O₂, selenium was eliminated from the peptide and a dimeric form of dimedone could be detected using LCMS and ¹H NMR. The dimeric dimedone product forms as a result of a seleno‐Pummerer reaction with Sec‐seleninic acid. Overall our results show that the reaction of dimedone with oxidized cysteine residues is quite different from the same reaction with oxidized selenocysteine residues.     

Iodination of p-nitrophenyl ethers used as photolabels

A system for radioactive labeling of compounds of biological interest that, due to their low electronic density, cannot be labeled by the standard iodination techniques is described. Using p-nitroanisole as a model, we have prepared 2-[¹²⁵I]iodo-4-nitroanisole by treatment with thallium trifluoroacetate, with later displacement of the thallium by iodide according to A. McKillop et al. (J. Amer. Chem. Soc.93, 4841–4844 (1970)). The labeled iodonitroanisole has been used as a photoactive reagent to label a protein (bovine serum albumin), showing that under the irradiation conditions used, the label is incorporated into the polypeptide mainly through modification of ?-amino groups of the lysine residues.