Membrane potential-dependent conformational changes in mitochondrially bound hexokinase of brain

Previously characterized monoclonal antibodies (Mabs) were used in a study of Type I hexokinase from rat brain. Based on the relative reactivity of these Mabs with soluble and mitochondrially bound forms, binding to mitochondria was shown to affect specific epitopic regions in both N- and C-terminal halves of the enzyme and to modulate conformational changes induced by binding of the ligands, Glc or ATP. Reactivities with Mabs recognizing epitopes in two defined regions of the N-terminal half and one defined region of the C-terminal half of the mitochondrially bound enzyme were selectively affected by mitochondrial membrane potential, or by addition of oligomycin, carboxyatractyloside, or bongkrekic acid. The Glc-6-P analog, 1 ,5-anhydroglucitol-6-P, was much more effective as a competitive inhibitor against extramitochondrial ATP than against intramitochondrial ATP generated by oxidative phosphorylation. These results provide further insight into the role of hexokinase-mitochondrial interactions in regulation of cerebral glucose metabolism.     

Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme

After denaturation in 0.6 M guanidine hydrochloride, rat brain hexokinase becomes highly susceptible to proteolysis by trypsin. Glucose 6-phosphate (Glc-6-P) and its analog, 1,5-anhydroglucitol 6-phosphate, selectively protect the N-terminal half of the molecule from proteolysis. These compounds do not protect the C-terminal half of the molecule, nor do they protect enzyme activity; the Glc analog, N-acetylglucosamine, does protect the C-terminal domain and catalytic activity, but does not prevent proteolysis of the N-terminal half of the molecule. These results are consistent with previous work [M. Nemat-Gorgani and J. E. Wilson (1986) Arch. Biochem. Biophys. 251, 97-103; D. M. Schirch and J. E. Wilson (1987) Arch. Biochem. Biophys. 254, 385-396] demonstrating that binding sites for both hexose and nucleotide substrates, and thus catalytic function, are associated with a 40-kDa domain located at the C-terminus of the enzyme. They further demonstrate that the binding site for the allosteric effector, Glc-6-P, lies in the N-terminal half of the molecule and is distinct from the catalytic site. Using protection against proteolysis as a reflection of binding, it is shown that the Glc-6-P binding site in the N-terminal region has all the characteristics described for the allosteric effector site on this enzyme in terms of affinity for Glc-6-P, specificity, and synergistic interactions with the hexose binding site in the C-terminal region of the molecule. This disposition of catalytic and regulatory functions in discrete halves of the molecule is consistent with suggestions by several investigators that mammalian hexokinases evolved by a process of duplication and fusion of an ancestral gene coding for a hexokinase similar to the present-day yeast enzyme, with the regulatory site of mammalian hexokinases having evolved from what was originally a catalytic site.     

Kinetic and regulatory properties of HK I(+), a modified form of the type I isozyme of mammalian hexokinase in which interactions between the N- and C-terminal halves have been disrupted

A modified form (HK I(+)) of rat Type I hexokinase (HK I) has been expressed. HK I(+) contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to alpha-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I(+) are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by P(i) result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I(+).     

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.     

Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain hexokinase has no preexisting allosteric site for glucose 6-phosphate

Difference spectroscopic investigations on the interaction of brain hexokinase with glucose and glucose 6-phosphate (Glc-6-P) show that the binary complexes E-glucose and E-Glc-6-P give very similar UV difference spectra. However, the spectrum of the ternary E-glucose-Glc-6-P complex differs markedly from the spectra of the binary complexes, but resembles that produced by the E-glucose-Pi complex. Direct binding studies of the interaction of Glc-6-P with brain hexokinase detect only a single high-affinity binding site for Glc-6-P (KD = 2.8 microM). In the ternary E-glucose-Glc-6-P complex, Glc-6-P has a much higher affinity for the enzyme (KD = 0.9 microM) and a single binding site. Ribose 5-phosphate displaces Glc-6-P from E-glucose-Glc-6-P only, but not from E-Glc-6-P complex. It also fails to displace glucose from E-glucose and E-glucose-Glc-6-P complexes. Scatchard plots of the binding of glucose to brain hexokinase reveal only a single binding site but show distinct evidence of positive cooperativity, which is abolished by Glc-6-P and Pi. These ligands, as well as ribose 5-phosphate, substantially increase the binding affinity of glucose for the enzyme. The spectral evidence, as well as the interactive nature of the sites binding glucose and phosphate-bearing ligands, lead us to conclude that an allosteric site for Glc-6-P of physiological relevance occurs on the enzyme only in the presence of glucose, as a common locus where Glc-6-P, Pi, and ribose 5-phosphate bind. In the absence of glucose, Glc-6-P binds to the enzyme at its active site with high affinity. We also discuss the possibility that, in the absence of glucose, Glc-6-P may still bind to the allosteric site, but with very low affinity, as has been observed in studies on the reverse hexokinase reaction.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

Functional interactions between the noncovalently associated N- and C-terminal halves of mammalian Type I hexokinase

The 100 kDa Type I isozyme of mammalian hexokinase has evolved by duplication and fusion of a gene encoding an ancestral 50 kDa hexokinase. Although the N- and C-terminal halves are similar in sequence, they differ in function, catalytic activity being associated only with the C-terminal half while the N-terminal half serves a regulatory role. The N- and C-terminal halves of rat Type I hexokinase have been coexpressed in M + R 42 cells. The halves associate noncovalently to produce a 100 kDa form that exhibits characteristics seen with the intact Type I isozyme but not with the isolated catalytic C-terminal half, i.e., characteristics that are influenced by interactions between the halves. These include a decreased K(m) for the substrate ATP and the ability of P(i) to antagonize inhibition by Glc-6-P or its analog, 1-5-anhydroglucitol-6-P. Thus, functional interactions between the N- and C-terminal halves do not require their covalent linkage.     

Study on ATP-generating system and related hexokinase activity in mitochondria isolated from undifferentiated or differentiated HT29 adenocarcinoma cells

The functional properties of mitochondria bound hexokinase are compared in two subpopulations of the HT29 human colon cancer cell-line: (1) the HT29 Glc+ cells, cultured in the presence of glucose, which are poorly differentiated and highly glycolytic and (2) the HT29 Glc- cells, adapted to grow in a glucose-free medium, which are 'enterocyte-like' differentiated and less glycolytic when given glucose (Zweibaum et al. (1985) J. Cell Physiol. 122, 21-28). The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase are found to be twice as high in Glc+ cells when compared to Glc- cells. Besides, the respiration rate is decreased in Glc+ cells compared to Glc- cells. These results correlate with the higher glycolytic rate in Glc+ cells. In many tissues, it has been shown that the binding of hexokinase to the mitochondrial outer membrane allows a preferential utilization of the ATP generated by oxidative phosphorylation which, in turn, is activated by immediate restitution of ADP. In highly glycolytic cancer cells, although a large fraction of hexokinase is bound to the mitochondria, the existence of such a channeling of nucleotides is still poorly documented. The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from both Glc+ and Glc- cells either with exogenous ATP or with ATP generated by mitochondria supplied with ADP and succinate (endogenous ATP). Diadenosine pentaphosphate (Ado2P5), oligomycin and carboxyatractyloside (CAT) were used in combination or separately as metabolic inhibitors of adenylate kinase, ATP synthase and ATP/ADP translocator, respectively. Exogenous ATP appears to be 6.5-times more efficient than endogenous ATP in supporting hexokinase activity in the mitochondria from Glc+ cells and only 1.8-times cells. The rate of oxidative phosphorylation being higher in mitochondria from Glc- cells, hexokinase activity is higher in this model when ATP is generated by respiration. Furthermore, in Glc+ mitochondria, the adenylate kinase reaction appears to be an important source of endogenous ATP for bound hexokinase, while, in Glc- mitochondria, hexokinase activity is almost totally dependent on the ATP generated by oxidative phosphorylation. This result might be explained by our previous finding that mitochondria from Glc+ cells lack contact sites between outer and inner membrane, whereas numerous contacts were observed in mitochondria from Glc- cells (Denis-Pouxviel et al. (1987) Biochim. Biophys. Acta 902, 335-348).     

Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme

A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.     

Sulfhydryl groups of rabbit liver arylsulfatase A

Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan residues. Chemical modification of the sulfhydryl groups does not alter the distinctive pH-dependent aggregation property of the arylsulfatase A. The free sulfhydryls of the enzyme react with numerous sulfhydryl reagents. Based on the reactions of iodoacetic acid, methyl methanethiosulfonate, 5,5'-dithiobis(2-nitrobenzoate) and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid with the sulfhydryl groups of arylsulfatase A, it is concluded that free sulfhydryls are not essential for the enzyme activity. In contrast, the observed inactivation of the enzyme by p-hydroxymercuribenzoate or p-hydroxymercuriphenylsulfonate is probably due to a modification of a histidine residue, consistent with previous reports that histidine is near the active site of arylsulfatase A. p-Hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate are able to react both with cysteine and with histidine residues of the protein molecule.     

Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half (mini-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 M). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 M). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the mini-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.     

Age-dependent changes in glucose 1,6-bisphosphate levels and in the activities of glucose 1,6-bisphosphatase, and particulate hexokinase and 6-phosphogluconate dehydrogenase in rat skin

The levels of glucose 1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, changed in rat skin during growth: Glc-1,6-P2 increased during the first week of age, and thereafter was dramatically reduced during maturation. The activity of glucose 1,6-bisphosphatase, the enzyme that degradates Glc-1,6-P2, changed with age in an invert manner as compared to the changes in Glc-1,6-P2. These findings suggest that the age dependent changes in this enzyme's activity may account for the changes in intracellular Glc-1,6-P2 concentration. The age-related changes in Glc-1,6-P2 were accompanied by concomitant changes in the activities of particulate (mitochondrial) hexokinase and 6-phosphogluconate dehydrogenase, the two enzymes known to be inhibited by Glc-1,6-P2. The activities of both these enzymes in the soluble fraction were not changed with age. The particulate enzymes were more susceptible to inhibition by Glc-1,6-P2 than the soluble activities, which may explain why only the particulate, but not the soluble activities, correlated with the age-dependent changes in tissue Glc-1,6-P2. These results suggest that the changes in particulate hexokinase and 6-phosphogluconate dehydrogenase resulted from changes in intracellular concentration of Glc-1,6-P2. The marked reduction in Glc-1,6-P2 during maturation, accompanied by activation of mitochondrial hexokinase and 6-phosphogluconate dehydrogenase, may reflect an enhancement in skin metabolism during growth.     

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

Differential response of cysteine residues in pig muscle phosphoglucose isomerase to seven sulfhydryl-modifying reagents

The three cysteine residues per subunit of pig muscle phosphoglucose isomerase show different reactivities toward various sulfhydryl reagents. The organomercurial, p-mercuribenzoate, can titrate two of the sulfhydryl groups under nondenaturing conditions. 2,2'-Dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, methyl 2-pyridyl disulfide, and 2-(2'-pyridylmercapto)mercuri-4-nitrophenol all label only one sulfhydryl group under the same conditions, whereas iodoacetic acid does not react with any of the sulfhydryl groups except when the enzyme is fully denatured. It is concluded, therefore, that charge, rather than steric restraint, is the determining factor for the differences seen in the modification patterns of the enzyme by these reagents. When enzyme was first labeled with 2,2'-dithiodipyridine and subsequently with p-mercuribenzoate, it was found that the latter, in a secondary process, will stoichiometrically react with the anion released by the former after the initial reaction with cysteine. The differences in reactivity of the cysteine residues toward the referred-to reagents have been exploited to specifically modify each of the three individual cysteine residues of pig muscle phosphoglucose isomerase.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

The mechanism of glucose 6-phosphate transport by Escherichia coli

To evaluate anion exchange as the mechanistic basis of sugar phosphate transport, natural and artificial membranes were used in studies of glucose 6-phosphate (Glc-6-P) and inorganic phosphate (Pi) accumulation by the uhpT-encoded protein (UhpT) of Escherichia coli. Experiments with intact cells demonstrated that UhpT catalyzed the neutral exchange of internal and external Pi, and work with everted as well as right-side-out membrane vesicles showed further that UhpT mediated the heterologous exchange of Pi and Glc-6-P. When loaded with Pi, but not when loaded with morpholinopropanesulfonate (MOPS), everted vesicles took up Glc-6-P to levels 100-fold above medium concentration in a reaction unaffected by the ionophores valinomycin, valinomycin plus nigericin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similarly, right-side-out vesicles were capable of Glc-6-P transport, but only if a suitable internal countersubstrate was available. Thus, in MOPS-loaded vesicles, oxidative metabolism established a proton-motive force that supported proline or Pi accumulation, but transport of Glc-6-P was found only if vesicles could accumulate Pi during a preincubation. After reconstitution of UhpT into proteoliposomes it was possible to show as well that the level of accumulation of Glc-6-P (17 to 560 nmol/mg of protein) was related directly to the internal concentration of Pi. These results are most easily understood if the transport of glucose 6-phosphate in E. coli occurs by anion exchange rather than by nH+/anion support.     

Yeast enolase carboxyl modification using Woodward's reagent K

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.