Activation of the latent human neutrophil gelatinase by urea.

The mechanism of activation of the latent human neutrophil gelatinase by urea has been studied in greater detail. After dialysis of the latent gelatinase against increasing concentrations of urea a considerable increase of its activity was observed. Moreover, the results indicate a progressive conversion of the latent 94,000 Da gelatinase into a proteolytically active fragment of 80,000 Da, which was subsequently processed to a few species of lower molecular mass inactive against gelatin. This conversion was completely inhibited by EDTA, suggesting an autocatalytic reaction. The inhibition was reversed by Zn2+ or Co2+. Thus, urea alters both the enzymatic and physical characteristics of the latent gelatinase which suggests that conformational changes may induce autoactivation of the latent enzyme.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.     

Partial purification and characterization of exudate gelatinase in the acute phase of carrageenin-induced inflammation in rats

Gelatinase has been partially purified from exudate in the acute phase of carrageenin-induced inflammation in rats. The enzyme occurs in a latent form that can be activated with 4-aminophenylmercuric acetate (APMA). The latent gelatinase was separated into an active gelatinase and a protein fraction by zinc-chelating Sepharose 6B column chromatography in the final step of purification, suggesting that the latent gelatinase is an enzyme-inhibitor complex. The pH optimum of the active gelatinase is about 7.5 and no reactivity toward native type I collagen or alpha-casein was detected. The molecular weights of the latent and active gelatinases were about 245,000 and about 185,000, respectively, as determined by gel filtration on Sephadex G-200. On the other hand, both latent and active gelatinases occurred in multiple forms in SDS-substrate polyacrylamide gel electrophoresis; the latent gelatinase showed two bands with molecular weights of 105,000 and 69,000, and two additional bands of 88,000 and 83,000 appeared when the latent gelatinase was activated with APMA, while the active gelatinase showed all four species. The active gelatinase was inhibited by metallo-proteinase inhibitors, but not by serine- or cysteine-proteinase inhibitors, suggesting that the exudate gelatinase is a metallo-proteinase. The active gelatinase was also inhibited by serum proteins such as albumin and gamma-globulin, suggesting that gelatinase does not remain in an active form in the inflammatory lesion, where the vascular permeability is increased.     

Further purification and some properties of a gelatin-specific proteinase of human leucocytes

A latent gelatin-specific proteinase (gelatinase) was isolated from the crude extract of human leukocytes. The enzyme was purified about 180-fold (7040 units/mg) with an overall yield of 23%. The isolated protein migrated as a single band on sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Its mobility was unaffected by reducing agent. The protein band corresponded to approx. 90-94 kDa. Gelatinase activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. This inhibition was reversed by Zn2+ and Co2+; other metal ions were less or not at all effective in reversing the inhibition. Moreover, Co2+ stimulated gelatinase activity. These results indicate that Zn2+ and/or Co2+ are essential for the activity of this gelatinase.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.     

Activation of human neutrophil gelatinase by endogenous serine proteinases.

The role of serine proteinases and oxidants in the activation of gelatinase released from human neutrophils was investigated. Gelatinase was measured by its ability to degrade both gelatin and native glomerular basement-membrane type IV collagen. When fMet-Leu-Phe or phorbol 12-myristate 13-acetate was used to stimulate the neutrophils, no gelatinase activity was measured in the absence of a mercurial activator, indicating that the enzyme was released entirely in latent form. However, when fMet-Leu-Phe-stimulated cells were treated with cytochalasin B, 50-70% of the maximal gelatinase activity was released. Activation was blocked by the serine-proteinase inhibitor phenylmethanesulphonyl fluoride and a specific inhibitor of neutrophil elastase, but was not affected by an inhibitor of cathepsin G. Addition of catalase or azide to prevent oxidative reactions did not affect activation of gelatinase under any conditions of stimulation, indicating that oxidants were not involved in activation. Our results imply that oxidative activation of gelatinase does not occur readily. However, neutrophil serine proteinases, particularly elastase, provide an alternative and apparently more efficient mechanism of activation.     

Purification to homogeneity of latent and active 58-kilodalton forms of human neutrophil collagenase

Latent and active 58-kDa forms of human neutrophil collagenase (HNC) have been purified to homogeneity. Buffy coats were extracted in the presence and absence of phenylmethanesulfonyl fluoride to generate crude starting preparations that contained latent and active HNC, respectively. The buffers used in preparing these extracts and for all subsequent chromatographic steps contained NaCl at a concentration of 0.5 M or greater, 0.05% Brij-35, concentrations of CaCl2 of 5 mM or greater, and (when feasible) 50 microM ZnSO4 to stabilize the HNC. The collagenase activity in the buffy coat extracts was adsorbed to a Reactive Red 120-agarose column at pH 7.5 in 0.5 M NaCl and was eluted when the NaCl concentration was increased to 1 M. The active and p-(chloromercuri)benzoate-activated latent enzymes were next adsorbed to a Sepharose-CH-Pro-Leu-Gly-NHOH affinity resin in 1 M NaCl at pH 7.5 and desorbed at pH 9 to give a fraction containing only HNC and a small amount of neutrophil gelatinase. The latter enzyme was removed by passage over a gelatin-Sepharose column in 1 M NaCl at pH 7.5. The purified samples of active and latent HNC were obtained with typical cumulative yields of 32 and 82% and specific activities toward soluble rat type I collagen at 30 degrees C of 7200 and 12,000 micrograms min-1 mg-1, respectively. These specific activities are markedly higher than previously reported for HNC. Both active and latent HNC exhibit a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the presence and in the absence of 2-mercaptoethanol. The mobility of latent HNC is consistent with a molecular weight of approximately 58K, with the active form exhibiting a slightly lower (less than 1-2K) molecular weight.     

Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM.

The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrading Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hydroxylase activity, whereas an inactive form accumulated in a recombinant strain. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by electrospray mass spectrometry, but nondenaturing gel filtration showed molecular masses of 31 600 Da for the inactive form and 11 500 Da for the active form. Cross-linking and sedimentation velocity results were consistent with the inactive form being a dimer. Partial thermal or chemical denaturation, or treatment with trifluoroethanol, readily activated dimeric DmpM. A combination of circular dichroism and fluorescence spectroscopies, activity assays, and native and urea gel electrophoresis were used to further characterize reactivation with urea. These results showed that dissociation of the dimeric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissociation also converts the monomeric form to a different conformation.     

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.     

Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase

Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.     

Cationic form of beta-galactosidase in the germinating seeds of Vigna sinensis (Linn) Savi

The cationic form of beta-galactosidase (EC 3.2.1.23) from the germinating seeds of Vigna sinensis has been separated from its other isoforms by DEAE-cellulose (DE-52) column chromatography and further purified by gel filtration and affinity chromatography. Polyacrylamide gel electrophoresis of the purified enzyme imparted a single protein band. The molecular mass of the enzyme as determined by Sephadex G-150 gel filtration is 58,800 Da. The optimum temperature and the optimum pH are 60 degrees C and 4.5, respectively. Most of the metal ions tested were inhibitory to the enzyme activity. The enzyme has Km for p-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-galactoside of 0.56 and 2.0 mM, respectively. The Ki values of galactose and lactose are 2.4 and 70.0 mM, respectively. The energy of activation of PNPG for the enzyme is 10.3 kcal/mol.     

Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms.

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.     

Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats

Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with a 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward alpha-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.     

Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Different effects of hypochlorous acid on human neutrophil metalloproteinases: activation of collagenase and inactivation of collagenase and gelatinase.

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.     

Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2.

A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.     

A dimer of a single polypeptide chain catalyzes the terminal four reactions of the L-tryptophan pathway in Euglena gracilis.

In Euglena gracilis the terminal four enzyme activities of the tryptophan biosynthetic pathway were found to be associated with a protein with an estimated molecular weight of 325,000 +/- 20,000. The protein was purified approximately 2,000-fold with relatively proportional recoveries of all four enzyme activities. The purified material was homogeneous by the criteria of analytical disc gel electrophoresis and gel isoelectric focusing. Disc gel electrophoresis after denaturation with sodium dodecyl sulfate gave a single protein band with a molecular weight of 155,000 +/- 5,000. Disc gel electrophoresis in 8 M urea also gave rise to a single protein band. We interpret these results as evidence for a single species of subunit. The pathway in Euglena is the only one known to the present in which the terminal enzyme, tryptophan synthase, is not a separate molecular species.     

Purification and properties of glucose-1-phosphate uridylyltransferase fromNeurospora crassa

A method was developed to assay glucose-1-phosphate uridylyltransferase and 2-acetamido-2-deoxyglucose-1-phosphate uridylyltransferase by separation and quantitation of the corresponding sugar nucleotides by HPLC. Glucose-1-phosphate uridylyltransferase (GPUT) fromNeurospora crassa was purified by a method involving ion-exchange, gel filtration, adsorption, and affinity chromatographic procedures. The enzyme was stable until the last step of purification, after which it became extremely labile, apparently due to disaggregation. With the purified enzyme, kinetic properties of GPUT were determined. Polyacrylamide gel electrophoresis (PAGE) of the purified enzyme under nondenaturing conditions showed a single band which contained all the enzymatic activity. Denaturation of the enzyme with sodium dodecyl sulfate followed by PAGE resolved the single band into four polypeptides of different molecular masses. The minimal molecular mass of the enzyme was calculated to be 537,000 Da. This value was similar to that calculated by sucrose density sedimentation, 580,000 Da, but different from that estimated by gel filtration, 1,600,000 Da. It is proposed that the native enzyme is a trimer which may be disaggregated. By electron microscopy of negatively stained samples, the enzyme appeared in the form of rosettes 10 nm in diameter.