Phylogeny and ontogeny of the phosphoglycerate mutases.--V. Inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. The three isozymes of glycerate-2,3-P2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P2 did not protect phosphoglycerate mutase against photoinactivation.     

Metabolism of glycerate-2,3-P2--IV. Effect of Hg2+ on the enzymes involved in the metabolism of glycerate-2,3-P2 in pig skeletal muscle

Type M phosphoglycerate mutase and skeletal muscle bisphosphoglycerate synthase-phosphatase from pig are similarly affected by Hg2+. Both enzymes lose the phosphoglycerate mutase and the glycerate-2,3-P2 synthase activities, and increase the glycerate-2,3-P2 phosphatase activity upon Hg2+-treatment. In contrast, bisphosphoglycerate phosphatase from pig skeletal muscle is inactivated by Hg2+. These results confirm the similarity between phosphoglycerate mutase and bisphosphoglycerate synthase-phosphatase. In addition they support the existence of separate binding sites for monophosphoglycerates and for bisphosphoglycerates at the phosphoglycerate mutase active site.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.     

Human erythrocyte phosphoglycerate mutase: Evidence for normal catalysis in the absence of added 2,3-bisphospho-D-glycerate

Kinetic analyses indicate that human erythrocyte phosphoglycerate mutase catalyzes the normal, reversible isomerization of D-glycerate-3-P and D-glycerate-2-P in the absence of added D-glycerate-2,3-P₂. The reaction is impeded, however, by a potent inhibitor which occurs as a natural component of commericial D-glycerate-3-P. Inhibition may be overcome through substrate purification or by adding D-glycerate-2,3-P₂ to the reaction medium containing the contaminant. In surmounting the inhibition, bisphosphoglycerate performs as a non-essential activator and not as a cofactor. The latter concept is corroborated by the observation that D-glycerate-2,3-P₂ has absolutely no influence on mutase catalysis conducted in the presence of pure substrate. The data presented here and elsewhere, however, suggest that the red cell enzyme is readily phosphorylated by mono- as well as bisphosphoglycerate. Additional findings show that at concentrations in excess of 3mM, D-glycerate-3-P accelerates phosphoglycerate mutase catalysis in the absence of cofactor, suggesting that the mutase molecule possesses a normal catalytic site and an allosteric activator site.     

Metabolism of glycerate 2,3-P2--XI. Essential amino acids of pig phosphoglycerate mutase isozymes

Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.     

The catalytic bimodality of mammalian phosphoglycerate mutase

Rabbit muscle phosphoglycerate mutase, presumed to manifest an absolute cofactor requirement for activity, has been found to express catalysis (3 +/- 1% of optimum) in the absence of added D-glycerate-2,3-P2. Isotope experiments indicate that this catalysis proceeds through a binary phosphoryl enzyme-glycerate intermediate which dissociates into free enzyme and monophosphoglycerate. 32P-Labeled phosphoglycerate mutase is formed by reaction with either D-32P-glycerate-3-P or D-U32P-glycerate-2,3-P2. In each case, the acid lability and alkali stability of the covalent adduct, phosphoenzyme, is consistent with a phosphohistidyl residue having been formed within the active site. D-[U-14C]Glycerate reacts with phosphoenzyme to generate D-[U-14C]monophosphoglycerate which, in turn, can react further with phosphoenzyme to yield D-[U-14C]glycerate-2,3-P2. The pH profile for the cofactor-independent activity exhibits an optimum at 6.0 as opposed to 7.0 when D-glycerate-2,3-P2 is present in the reaction medium. Bisubstrate kinetics (pH 7.0, 23 degrees C) with D-glycerate-3-P concentration as the variable, yields a family of reciprocal plots which is in accord with a modified ping-pong mechanism when D-glycerate-2,3-P2 concentrations are greater than 10(-1) Km (where Km = 0.33 microM). Progressively diminishing concentrations (much less than Km) of D-glycerate-2,3-P2 produce curvilinear reciprocal plots that approach linearity as a limit in accordance with single substrate kinetics.     

Rates of phosphorylation and dephosphorylation of phosphoglycerate mutase and bisphosphoglycerate synthase.

Phosphoglycerate mutase and bisphosphoglycerate synthase (mutase) can both be phosphorylated by either glycerate-1,3-P2 or glycerate-2,3-P2 to form phosphohistidine enzymes. The present study uses a rapid quench procedure to determine if, for each enzyme, the formation of the phosphorylated enzyme and phosphate transfer from the enzyme can occur at rates consistent with the overall reactions. With bisphosphoglycerate synthase from horse red blood cells (glycerate-1,3-P2 leads to glycerate-2,3-P2) at pH 7.5, 25 degrees, phosphorylation of the enzyme appears rate-limiting, k = 13.5 s-1, compared with kcat = 12.5 s-1 for the overall synthase rate. Phosphoryl transfer from the enzyme to phosphoglycerate occurs at 38 s-1 at 4 degrees and was too fast to measure at 25 degrees. With chicken muscle phosphoglycerate mutase the half-times were too short to measure under optimal conditions. The rate of enzyme phosphorylation by glycerate-2,3-P2 at pH 5.5, 4 degrees, could account for the overall reaction rate of 170 s-1. The rate of phosphoryl transfer from the enzyme to glycerate-3-P was too rapid to measure under the same conditions. It is concluded that the phosphorylated enzymes have kinetic properties consistent with their participation as intermediates in the reactions catalyzed by these enzymes.     

Purification and characterization of phosphoglycerate mutase isozymes from pig heart

The three isozymes of phosphoglycerate mutase from pig heart have been purified to homogeneity. The isozymes have a molecular weight of 57000 as determined by gel-filtration chromatography. Discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate yields a single band with a molecular weight of 29000, indicating that the isozymes are dimers composed of subunits of similar mass. Hybridization experiments show that the three isozymes result from homodimeric and heterodimeric combinations of two different subunits. The two types of subunit differ in their heat lability and in the presence of -SH groups essential for enzymatic activity. No remarkable differences exist in the kinetic constants of the purified isozymes. The kinetic pattern is consistent with a 'ping-pong' mechanism. The homogeneous preparations of the three isozymes show intrinsic glycerate-2,3-P2 synthase activity and glycerate-2,3-P2 phosphatase activity which can be stimulated by glycolate-2-P.     

Immunological properties of rat phosphoglycerate mutase isozymes

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.     

Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle

Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite).     

Purification, characterization and immunological properties of 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize (Zea mays) seeds

2,3-Bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) was purified and characterized from maize. SDS electrophoresis showed only one band with a molecular mass of 64 kDa, similar to that determined for the native enzyme by gel-filtration chromatography. The kinetic constants were similar to those reported for wheat germ phosphoglycerate mutase. Rabbit antiserum against maize phosphoglycerate mutase possesses a high degree of specificity. It also reacts with the wheat germ enzyme but fails to react with other cofactor-independent or cofactor-dependent phosphoglycerate mutases. Cell-free synthesis experiments indicate that phosphoglycerate mutase from maize is not post-translationally modified.     

Hybrid forms of phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase-phosphatase

Purified phosphoglycerate mutase from pig skeletal muscle and 2,3-bisphosphoglycerate synthase-phosphatase from pig erythrocytes were hybridized “in vitro”. The hybrid showed a behaviour on electrophoresis and on ion-exchange chromatography similar to that of a naturally occurring enzyme with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities present in pig skeletal and heart muscle. Both the hybrid and the muscle enzyme possess similar activities ratio. From these and previous data it is suggested that the six enzymatic forms with phosphoglycerate mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities detected in mammalian tissues (Carreras et al. 1981, Comp. Biochem. Physiol. 70B, 477–485) result from combination of three subunits (types M, B and E).     

Active site phosphohistidine peptides from red cell bisphosphoglycerate synthase and yeast phosphoglycerate mutase.

Bisphosphoglycerate synthase (glycerate-1,3-P2 yields glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P formed from glycerate-2-P) are both phosphorylated by substrates at a histidine residue forming covalent intermediates which have been shown to function in the phosphoryl transfer reactions catalyzed by these enzymes (Rose, Z. B., and Dube, S. (1976) J. Biol. Chem. 251, 4817--4822). We have phosphorylated bisphosphoglycerate synthase from horse red blood cells with [U-32P]glycerate-2,3-P2, digested with trypsin, and purified the phosphopeptide. The amino acid sequence of the phosphohistidine peptide has been determined to be: His-Gly-Gln-Gly-Ala-Trp-Asn-Lys. In like manner, a phosphohistidyl peptide has now been purified from yeast phosphoglycerate mutase, for which the amino acid sequence is known (Winn, S. I., Watson, H. C., Fothergill, L. A., and Harkins, R. N. (1977) Biochem. Soc. Trans. 5, 657-659). The amino acid composition of the phosphopeptide indicates that histidine-8 was phosphorylated. The sequence of this peptide is closely homologous with the active site peptide from bisphosphoglycerate synthase. In yeast phosphoglycerate mutase, the denatured phosphoenzyme hydrolyzes with a single rate constant of 2.02 X 10(-4) s-1 at pH 3, 45 degrees C. The relevance of these observations to the enzymatic mechanism is discussed.     

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.     

Phosphoglycerate mutase. Kinetics and effects of salts on the mutase and bisphosphoglycerate phosphatase activities of the enzyme from chicken breast muscle.

The steady state kinetics and effects of salts on chicken breast phosphoglycerate mutase have been examined. The enzyme can catalyze three phosphoryl transfer reactions: mutase, bisphosphoglycerate phosphatase, and bisphosphoglycerate synthase. The mutase rate was measured in the favorable direction (Keq = glycerate-3-P/glycerate-2-P approximately equal to 12) using [2T]glycerate-2-P as substrate. The bisphosphoglycerate phosphatase activity was studied in the presence of the activator, glycolate-2-P. The latter is an analog of the glycerate-P's and appears to act as an abortive mutase substrate. The kinetic pattern obtained with both activities is that of a ping-pong mechanism with inhibition by the second substrate occurring at a lower concentration than the Km value for that substrate. The kinetic parameters for the mutase determined in 50 mM N-[tris(hydroxymethyl)methyl-2-amino]ethanesulfonate (TES)/sodium buffer containing 0.1 M KCl, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.069 micron; Km glycerate-2-P, 14 micron; Km glycerate-3-P approximately 200 micron; Ki glycerate-2-P, 4 micron. The kinetic parameters for the phosphatase reaction in 50 mM triethanolamine/Cl- buffer, pH 7.5, 25 degrees C are: Km glycerate-2,3-P2, 0.065 micron:Km glycolate-2P, 479 micron; Ki glycolate-2-P, 135 micron. The enzyme is sensitive to changes in the ionic environment. Increasing salt concentrations activate the phosphatase in the presence of glycolate-2-P by decreasing the apparent Km of glycerate-2,3-P2. The effects are due to the anionic component and Cl- greater than acetate greater than TES. The same salts are competitive inhibitors with respect to glycolate-2-P. With high levels of KCl that produce a 30-fold decrease in the apparent maximal velocity due to competition with glycolate-2-P, the Km of glycerate-2,3-P2 remains low. These observations lead us to postulate that each monophosphoglycerate substrate has a separate site on the enzyme and that glycerate-2,3-P2 can bind to either site. The binding of anions to one site of the nonphosphorylated enzyme allows an increase in the on and off rates of glycerate-2,3-P2 at the alternate site. Salts inhibit the mutase reaction. The Km of glycerate-2,3-P2 is increased as is that of glycerate-2-P. The effect on the Km of glycerate-2,3-P2 is attributed to an increase in the off rate/on rate ratio for glycerate-2,3-P2. The bisphosphoglycerate synthase reaction is shown to require added glycerate-3-P. The equilibrium between enzyme and glycerate-1,3-P2 is favorable (Kdiss less than or equal 7 X 10(-8) M) and suggests that in the absence of a separate synthase this reaction may have functional significance.     

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

Purification and properties of the phosphoglycerate mutase isozymes from the mouse

The two homodimeric isozymes of phosphoglycerate mutase have been purified from murine kidney and muscle. No differences were observed in the Michaelis-Menten constant for the substrate 2-phospho-D-glycerate, in molecular weight, temperature and pH optima, when the purified isozymes were compared. The isozymes differ in their inhibition constants for phosphoenolpyruvate, in their Michaelis constants for 3-phospho-D-glycerate and 2,3-bisphospho-D-glycerate, their thermal and pH lability and in their sensitivity towards mercury ions.     

Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.

Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1).     

Lily cofactor-independent phosphoglycerate mutase: purification, partial sequencing, and immunolocalization

A cofactor-independent phosphoglycerate mutase (PGAM-i) was isolated to homogeneity from monocotyledonous Lilium longiflorum Thunb. Two-dimensional (2D) polyacrylamide gel electrophoresis resolved three PGAM-i forms. This enzyme was originally identified by cross-reactivity to antibodies affinity-purified from 2D gels using human vitronectin (VN). Antibody produced against a denatured protein spot from a 2D gel did not recognize VN protein, but partial protein and DNA sequencing showed similarity of the former protein to maize PGAM-i. Immunoblots from roots, styles, leaves, and anthers showed the presence of PGAM-i in all tissues examined; it was isolated predominantly from the soluble cell fraction, with some present in the insoluble cell fraction. Immunoelectron microscopy demonstrated its localization in the cytoplasm and plastids in root cells near the apical meristem. In addition, immunogold labeling detected signals from the nucleus. The immunohistochemical localization of the enzyme in the nucleus, as well as in the cytosol and plastids, indicates that lily PGAM-i might have multiple functions in the cell.Abbreviations ELISA enzyme-linked immunosorbent assay - PGAM cofactor-dependent phosphoglycerate mutase - PGAM-i cofactor independent phosphoglycerate mutase - TEM transmission electron microscopy - 2D two dimensional - VN vitronectin