Striking differences of physical properties for two different phenylalanyl-tRNA synthetases in Cyanophora paradoxa

Abstract Extracts of Cyanophora paradoxa contain 2 activities for phenylalanyl-tRNA synthetase (PRS), when assayed with yeast and eubacterial tRNA as substrate. These enzymes can be separated by salting-out chromatography. Subsequent gel filtration revealed a striking difference in M _r between the 2 enzymes. Whereas the M _r of the enzyme aminoacylating yeast tRNA is 260 000, typical for PRS from lower eukaryotic organisms, the second enzyme has a unique M _r of 80 000. It aminoacylates eubacterial tRNA, but shows no immunological relationship to the corresponding enzyme from Escherichia coli .     

Purification and characterization of type I DNA topoisomerase from Lentinus edodes

Abstract Type I DNA topoisomerase was purified from the lower eukaryote Lentinus edodes . Like the topoisomerase I from other eukaryotic cells, the L. edodes enzyme removed both positive and negative superhelical turns. The M _r of the enzyme was determined to be 71,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On gel filtration by Sephacryl S-200, the enzyme appeared to be an aggregate with a native M _r of about 235 000 daltons. No energy cofactor was required and ATP did not affect the enzyme. Activity was enhanced about 10-fold by Mg²⁺ (10 mM) and about 8-fold by KCl (100 mM).     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

Molecular masses of cAMP-binding proteins in yeasts

Abstract The cAMP-binding proteins of different yeasts were photoaffinity labeled using 8- N ₃-[³²P]cAMP, and the M _r values of the labeled proteins estimated by SDS-polyacrylamide gel electrophoresis. The M _r values of the cAMP-binding proteins may be grouped into two size classes: (A) M _r of about 50 000 represented by Saccharomyces cerevisiae and S. uvarum , and (B) M _r of about 60 000 represented by Kluyveromyces fragilis, K. lactis, K. marxianus, S. globosus and S. rouxii .     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

Ornithine carbamoyltransferase of Streptomyces fradiae: purification and properties

Abstract The enzyme ornithine carbamoyltransferase was purified from Streptomyces fradiae . A 1200-fold increase in specific activity was achieved by ammonium sulphate precipitation, DEAE-cellulose and aminohexyl-agarose chromatography and gel filtration. The purified enzyme has a M _r of 87 000. Its isoelectric point is 5.3 as determined by isoelectric focusing. Apparent K _m values at pH 7.7 for ornithine and carbamoyl phosphate are 1.8 and 1.2 mM, respectively.     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum

Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton rubrum by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000- M _r (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15,000- M _r (reduced SDS-PAGE) and 37,000- M _r (substrate SDS-PAGE). It had a pH optimum of 7.5 and a p I of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000- M _r proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides immitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinase to be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the proteinase remains speculative although it may play a role in the development and subsequent proliferation of the fungus in vivo.     

Localisation of the glucose-fructose oxidoreductase in wild type and overproducing strains of Zymomonas mobilis

Abstract The enzyme glucose-fructose oxidoreductase (GFOR) from the Gram-negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of M _r 42 500. Using immunogold-labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6-fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre-GFOR protein ( M _r 48 000).     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Ring dechlorination of deethylsimazine by hydrolases from Rhodococcus corallinus

Abstract Cell-free extracts of Rhodococcus corallinus NRRL B-15444R dechlorinated deethylsimazine (2-chloro-4-ethylamino-1,3,5-triazine-6-amine) to N -ethylammeline (2-amino-4-ethylamino-1,3,5-triazine-6(5H)-one) anaerobically, and, thus, presumably hydrolytically. Two enzyme fractions ( M _r about 450 000 and 180 000) that dechlorinated deethylsimazine were detected after gel permeation chromatography of cell extracts.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Purification of aspartate transcarbamoylase from Pseudomonas syringae

Abstract The aspartate transcarbamoylase (ATCase) from Pseudomonas syringae has been purified. The purified enzyme was shown by SDS-PAGE to give two bands. Unambiguous results from N-terminal sequencing suggested that each band represented a homogeneous polypeptide. The M _r (relative molecular mass) of the polypeptides was estimated to be 47 kDa and 34 kDa. The M _r of the holoenzyme determined by gel filtration and electrophoretic migration in polyacrylamide gradient gels under non-denaturing conditions was estimated at approximately 490 kDa. These findings suggest a subunit structure different from any previously described for a bacterial ATCase.     

Methods for the identification and analysis of l-altrose in bacterial polysaccharides

Abstract The two genes ( cfxP ) for phosphoribulokinase (PRK) in Alcaligenes eutrophus H16 are simultaneously expressed, resulting in the formation of PRK isoenzymes. The isoenzymes are structurally and immunoligically closely related. Their subunits differ only slightly in size. M _rs of 33 000 and 32 500 were determined for the chromosomally and megaplasmid pHF1-encoded subunits, respectively. The pHG1-encoded gene, cfxP , was cloned in Eschirichia coli and expressed under the cloned in Escherichia coli and expressed under the control of the lac promoter of pUC9 vectors. Native PRK with subunits of M _r 32 500 was formed, confirming the identity and functionality of cfxP _p . However, the recombinant PRK had a significantly lower specific activity than the authentic enzyme.     

Two different genes and gene products for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCOase) in Nitrobacter hamburgensis

Abstract Heterologous DNA hybridization using a ribulose- 1,5-biphosphate carboxylase/oxygenase (RuBisCOase) large subunit gene ( rbc L) probe from Anacystis nidulans revealed the presence of two rbc L in Nitrobacter hamburgensis . One gene is located on a plasmid, the other on the chromosome. The genes appear to be very similar since both hybridized strongly to the A. nidulans probe. However, restriction endonuclease digestions revealed differences.
Two different RuBisCOase enzymes were isolated from N. hamburgensis. The M _r of the native enzymes were 520 000 and 480 000. Sodium dodecyl sulfate-polycrylamide gel electrophoresis (SDS-PAGE) revealed the presence of both LSU and small subunits (SSU) for both enzymes. The M _r were 53 000 and 16 000, and 49 000 and 13 500, respectively. A hexadecameric structure is suggested for both enzymes.     

Intracellular precursors of endo-β-1,4-glucanase in Trichoderma reesei

Abstract Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting was employed to detect intracellular precursors of endo-β-1,4-glucanases (EGs) in Trichoderma reesei QM9414 under conditions of de novo induction by sophorose and de novo carbon catabolite derepression by lactose. Secretion of EGs was always preceded by intracellular accumulation of lower M _r precursors, which became processed to larger M _r forms immediately prior to their extracellular appearance. Treatment of the larger M _r forms with α-mannosidase converted them to forms with the same M _r as the smaller forms, whereas Endo H treatment was without effect. These results are consistent with a requirement of O -linked glycosylation for secretion of EGs by T. reesei .