Physicochemical properties of a beta-lactamase from Streptomyces UCSM-104

A purified beta-lactamase from Streptomyces UCSM-104 shows the presence of three subforms when stained for protein and/or for activity after polyacrylamide gel electrophoresis or after electrofocusing. The pI values of the three subforms were 5.45, 5.30 and 5.10, respectively. The respective electrophoretic mobilities were 4.6 X 10(-5), 5.2 X 10(-5) and 5.9 X 10(-5)m2/sV. Relative molecular mass of 14,900 was determined. The amino acid composition was established. Cysteine was not detected. A fairly high proline content (8.3%) differentiates this enzyme from other beta-lactamases. Lysine was the only N-terminal amino acid detected after dansylation. The possible origin of the subforms is discussed.     

Heterogeneity of liver alcohol dehydrogenase on starch-gel electrophoresis

1. Purified horse-liver alcohol dehydrogenase is heterogeneous on starch-gel electrophoresis in several buffer systems. 2. The electrophoretic pattern is altered by the addition to the buffers of oxidized or reduced coenzymes, isobutyramide, metal ions or metal-chelating agents. 3. The effect of coenzymes on the pattern suggests that the major cause of the observed heterogeneity is not the existence of isoenzymes, but the presence in the enzyme preparations of coenzyme-enzyme complexes or complexes with other nucleotides similar to, but less reactive than, the coenzymes. 4. Metal ions and chelating agents influence the electrophoretic separation by partial denaturation and inactivation of the enzyme.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.     

Purification and characterization of human prostatic acid phosphatase.

Human prostatic acid phosphatase (orthophosphoric monoester phosphohydrase, EC 3.1.3.2) is purified to homogeneity by standard procedures which include CM-Sephadex, Con A affinity chromatography and gel filtration. The purified enzyme is antigenically specific and has a M.W. of 100,000 with subunit M.W. of 48,000. However, the enzyme exhibited charge heterogeneity. Two major electrophoretic or chromatographic isozymic forms of PAP were separated by DEAE-Sephadex chromatography and their immunochemical identity was studied by immunodiffusion before and after the neuraminidase digestion. Quantitative precipitin and inhibition experiments showed immunological identity of the two chromatographic isozymes. Immunologic specificity of this enzyme resides on the protein moiety rather than the carbohydrate residue, although the latter group is mostly responsible for the charge group heterogeneity of the enzyme.     

Separation of the estrogen-activated growth-regulatory forms of alpha-fetoprotein in mouse amniotic fluid.

We have previously reported that murine fetal alpha-fetoprotein (AFP) incubated for 1.0 h at room temperature in the presence of high concentrations of estradiol (E2) generates a growth-regulatory product designated AFP/E2. Subsequently we developed a bioassay in the immature mouse uterus to measure both the growth-inhibitory and growth-enhancing properties of AFP. In the present study, we have employed this bioassay to monitor each of the amniotic fluid-derived AFP isolates fractionated by various chromatographic and electrophoretic techniques. The objective of this investigation was to partition and isolate the various molecular forms of AFP contained in amniotic fluid and determine whether the growth-regulatory activities resided with one or more of the fractions. AFP was fractionated by three different chromatographic/electrophoretic methods: E2 affinity chromatography, preparative polyacrylamide-gel electrophoresis (PAGE), and high-performance liquid chromatography (HPLC); and one immunoaffinity method: gel-entrapped antibody filtration (GAF). Whereas E2 affinity chromatography separated the biological activity of AFP into inhibitory and possibly enhancing activities, PAGE purification yielded three fractions: an inhibitor, an enhancer, and a fraction without growth-regulatory activity. Immunoaffinity separation yielded an AFP product with only inhibitory activities. In comparison, fractionation by HPLC produced seven AFP fractions in which only three displayed growth-regulatory activities: two inhibitory and one enhancing. After subsequent HPLC rechromatography of these fractions, none displayed any biological activity. Thus, murine AFP derived from amniotic fluid is composed of potential heterofunctional forms that, depending on their relative abundance in the preparation, constitute a mixture capable of either (a) growth inhibition, (b) no effect, or (c) growth enhancement.     

Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and beta-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.     

Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.     

Expression,purification, and characterization of the oligomeric states of rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum

The rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum was successfully overexpressed in Escherichia coli with 6x His-Tag and purified by using Ni-NTA technology. It was characterized by SDS and native polyacrylamide gel electrophoresis (PAGE), as well as size-exclusion chromatography. The protein was pure, judged by SDS-PAGE, but three or more oligomeric species were observed by native PAGE and size-exclusion chromatography. The smallest rubredoxin oxidoreductase species is the dimer. The multiple species are stable and remain in their respective oligomeric states, judged by the chromatographic and electrophoretic results. A model is proposed in order to explain the structural basis for these results.     

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.     

Effect of Size,Quaternary Structure and Translational Error on the Static and Dynamic Heterogeneity of β-Galactosidase and Measurement of Electrophoretic Dynamic Heterogeneity

Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on β-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli β-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.     

Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydrolyase from stroma of spinach chloroplasts

The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction.

An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their K_m and pH optima differing only very slightly. Response to some metabolites is reported.

     

Transformation and laccase mutant isolation in Coprinus congregatus by restriction enzyme-mediated integration

Coprinus congregatus did not show any growth on a minimal medium in the presence of phosphinothricin which inhibited glutamine synthetase. Genetic transformation to phosphinothricin resistance in C. congregatus was carried out successfully by restriction enzyme-mediated integration. The procedure was improved to yield 550 transformants per μg of DNA, and three laccase mutants were generated. The vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from the transformants by Southern hybridization. Transformants showed identical electrophoretic banding patterns but CL1430b had a faster moving band when analyzed by native PAGE.     

Intact form of myeloperoxidase from normal human neutrophils

Myeloperoxidase (donor: H₂O₂ oxidoreductase, EC 1.11.1.7) of human polymorphonuclear neutrophils was purified rapidly in the presence of the protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The purified enzyme behaved as a single molecular species in several nondenaturing electrophoretic and chromatographic systems. Peroxidase activity in fresh extracts of neutrophils from 20 normal persons and from 5 patients with polycythemia was electrophoretically identical to purified enzyme. Treatment with trypsin converted myeloperoxidase to multiple electrophoretic forms of active enzyme. Size (M_r ca. 15,000 and ca. 55,000) and stoichiometry of the subunits of purified enzyme, and enzyme M_r ca. 140,000, were compatible with intact myeloperoxidase having an α₂β₂ structure. We found no evidence for electrophoretically detectable genetic polymorphism of myeloperoxidase. Proteolytic degradation of myeloperoxidase probably accounted for electrophoretic heterogeneity of enzyme and for some constituent peptides described previously.     

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosol deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey, and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. These findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybrid cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed. The chick-mouse hybrid cells contained cytosol dCMP deaminase activity, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.This investigation was aided by Grant Q-163 from the Robert A. Welch Foundation and by USPHS Grants CA-06656-12 and 1-K6-AI 2352 from the National Cancer Institute and the National Institute of Allergy and Infectious Diseases.     

Quantitation of aspartate aminotransferase isoenzymes after electrophoretic separation

A scheme for the quantitative detection of aspartate aminotransferase isoenzymes and multiple forms after electrophoretic separation is described. Glutamate generated from the aminotransferase reaction is quantitated by using the glutamate dehydrogenase/diaphorase-coupled enzyme system to form a formazan dye. Product inhibition of aspartate aminotransferase by oxaloacetate is prevented by including oxaloacetate decarboxylase in the overlay reagent. Results compare favorably with those of an immunochemical precipitation procedure. The method can also be used to detect quantitatively subforms and atypical forms (genetic variants, immunoglobulin-enzyme complexes) of aspartate aminotransferase.     

Purification and partial characterization of the hydroxylase component of the methanesulfonic acid mono-oxygenase from methylosulfonomonas methylovora strain M2.

The reductase enzyme and the hydroxylase enzyme of the three-component methanesulfonic acid mono-oxygenase (MSAMO) from Methylosulfonomonas methylovora were purified. Purification of the reductase from M. methylovora using a range of chromatographic techniques was accompanied by complete loss of activity. Expression of the reductase as a glutathionine S-transferase fusion protein in Escherichia coli cells was successful as judged from the size of the polypeptide band obtained on induction with isopropyl thio-beta-D-galactoside. Subsequent affinity purification of the fusion protein, however, led to a protein extract containing only glutathionine S-transferase protein, indicating that the fusion protein was unstable in vitro. The hydroxylase component of the MSAMO was purified from M. methylovora to near electrophoretic homogeneity using Q-Sepharose, hydroxyapatite and Mono Q chromatography. SDS/PAGE of the purified hydroxylase showed a single band at approximately 43.7 kDa for the alpha-subunit and a double band at approximately 23 kDa for the beta-subunit. MS scans obtained with matrix-assisted laser desorption/ionization and electrospray ionization showed single peaks for both subunits, with a mass of 48 145.4 Da for alpha, 20 479.1 Da for beta, and 68 624.5 for the alphabeta-monomer. Gel filtration revealed a mass of 209 kDa, suggesting an alpha3beta3 structure for the native enzyme. Purified hydroxylase enzyme exhibited absorbance maxima at 330 nm, 460 nm and 570 nm, indicating the presence of iron-sulfur centres. The protein preparations contained 1 mol sulfide and 3-4 mol iron per mol alphabeta-monomer. Chromium, cobalt, copper, lead, nickel, molybdenum, tungsten and vanadium were not found. Flavins were also absent. Antibodies raised against the native hydroxylase enzyme cross-reacted with cell-free extract from M. methylovora cells grown with methanesulfonate, but not with extract from cells grown with methanol, confirming that MSAMO was specifically induced during growth on methanesulfonate.     

Electrophoretic properties of ovomucoid

1. The nature of the electrophoretic heterogeneity of ovomucoid was investigated. Optimum resolution of the fractions on starch-gel electrophoresis occurred over a narrow range of pH and ionic strength. The pattern was not altered in the presence of 8m-urea but the bands were sharper. Ovomucoid–trypsin complex is stable at pH4·6 but dissociated in 6m-urea. 2. The two major fractions of ovomucoid were eluted from the gels. One of these was virtually free of sialic acid and the other, which contained 0·4mole of sialic acid/mole of protein, split into two components on electrophoresis after neuraminidase treatment. It was concluded that these two components, and likewise the two major fractions of ovomucoid, differ by a unit charge/mol. Differences in sialic acid content account for only part of the electrophoretic heterogeneity of ovomucoid.     

The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae.

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloramphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight from of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.