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PubMedDate not supplied

[Enzymes incorporated in polyelectrolyte complexes. Effect of matrix conformational changes and phase transitions in solutions on catalytic properties].

Immobilization of enzymes (penicillin amidase and alpha-chymotrypsin) in water-soluble nonstoichiometric polyeloctrolyte complexes (PEC) formed by poly(4-vinyl-N-ethylpyridinium bromide) (polycation) and polymethacrylic acid (polyanion) was carried out. Particles of these PEC consist of a nucleus formed by sequences of salt bonds between the units of oppositely charged polyelectrolytes and the hydrophylic shell formed by ionized groups of polyanions which is in excess in PEC. Such a structure of PEC particles results in a cooperative phase transitions of these systems at slight variations of pH and ionic strength. The work demonstrates phase diagrams of PEC solutions. The values of pH and ionic strength at which phase transitions in solutions of different PEC occur were elucidated. The decrease of pH value from 6.1 to 5.7 leads to reversible phase transition followed by a saltatory increase of Km for immobilized penicillin amidase by 5-10 fold depending on substrate used. The phase transition induced by ionic strength increase up to 0,27 M NaCl doesn't change significantly the Km-value of enzymic reaction. The phenomenon observed can be accounted for by the different structure of PEC particles. The catalytic properties of immobilized chymotrypsin were shown to depend on the loci of enzyme attachment. If the enzyme is bound to polyanion, neither conformational changes of the matrix nor phase transition in solution influence its accessibility for the protein inhibitor, but rather change the binding constant. If the enzyme is attached to polycation, i.e. included in the polycomplex nucleus, two fractions of enzymes accessible and inaccessible for protein inhibitor appear.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin↗

PubMed1984

An identical cytochrome b561 is present in bovine adrenal chromaffin vesicles and posterior pituitary neurosecretory vesicles.

An identical cytochrome b561 was found to be an integral component of both chromaffin vesicles from adrenal medulla and neurosecretory vesicles from posterior pituitary by spectrophotometric and immunological techniques. The neurosecretory vesicles had 6.8 micrograms of cytochrome/mg of membrane protein versus 69 micrograms/mg in chromaffin vesicles. This cytochrome was also immunologically detected in various regions of bovine brain and was immunologically distinct from the cytochrome found in serotonin-containing vesicles from platelets. Dopamine beta-hydroxylase involved in the biosynthesis of catecholamines was not present in neurosecretory vesicles, suggesting an alternative functional role for the cytochrome in these vesicles. Neurosecretory vesicles do contain a mixed function oxidase (peptidyl alpha-amidase) which appears to be involved in alpha-amidation of the carboxyl termini of vasopressin and oxytocin. We suggest that cytochrome b561 in the two vesicles may be functionally associated with different ascorbic acid-dependent, copper-containing mixed function oxidases: dopamine beta-hydroxylase and peptidyl alpha-amidase.

Adrenal Medulla↗

PubMed1980

Linear, uncross-linked peptidoglycan secreted by penicillin-treated Bacillus subtilis. Isolation and characterization as a substrate for penicillin-sensitive D-alanine carboxypeptidases.

Incubation of growing Bacillus subtilis with penicillin G led to the secretion of a peptidoglycan-related polymer and a nonglycan-bound pentapeptide into the culture medium. The secreted polymer was isolated and characterized as a linear cell wall glycan strand substituted predominantly by uncross-linked pentapeptide side chains. Polymer formation and secretion are most likely the result of continued synthesis and elongation of nascent glycan strands in the absence of subsequent processing by peptidoglycan transpeptidase or D-alanine carboxypeptidase enzymes. The nonglycan-bound pentapeptide, L-Ala-D-iso-Glu-meso-diaminopimelic acid-D-Ala-D-Ala, was probably formed by an N-acetylmuramyl-L-alanine amidase active on the peptide side chains of The uncross-linked polymer. The uncross-linked peptidoglycan polymer was shown to be a good substrate for penicillin-sensitive D-alanine carboxypeptidases purified from membranes of B. subtilis, Bacillus stearothermophilus, and Escherichia coli. D-alanine release was not, however, coupled to the cross-linking of peptide side chains, suggesting that these enzymes do not function as peptidoglycan transpeptidases in vivo. No transpeptidase or D-alanine carboxypeptidase activity was detected in mixtures of high molecular weight penicillin-binding proteins from B. subtilis, B. stearothermophilus, or Staphylococcus aureus. Possible reasons for the inability to demonstrate these activities are discussed. In addition, an N-acetylmuramyl-L-alanine amidase activity which copurifies with penicillin-binding proteins from B. subtilis, S. aureus, and E. coli was partially characterized.

Amino Acids↗

PubMed1982

Biosynthesis of S-methyl-N-oleoylmercaptoethylamide from oleoyl coenzyme A and S-adenosylmethionine.

Addition of oleoyl-CoA to incubations containing rat lung membranes and S-adenosyl [methyl-3H]methionine resulted in the formation of a previously unidentified nonpolar methylated lipid. The product was formed enzymatically, with an apparent Km for S-adenosylmethionine (AdoMet) of about 0.3 microM and half-maximal activity using about 0.1 mM oleoyl-CoA. Activity was highest in microsomes but present in other membranous fractions, including plasma membranes from mature human erythrocytes. Intact red blood cells formed the nonpolar methylated lipid intracellularly upon incubation with [methyl-3H]methionine and oleoyl-CoA. Product formation differed among membranes from various tissues. The nonpolar methylated lipid was analyzed by TLC, high performance liquid chromatography, and gas chromatography with radiodetection. It was identified as S-methyl-N-oleoylmercaptoethylamide by gas chromatography-mass spectrometry. Products obtained from oleoyl-CoA or palmitoyl-CoA, incubated with nonradioactive or [methyl-14C]AdoMet, were compared using electron impact and/or chemical ionization mass spectrometry. Inferred structures were confirmed using authentic standards. The methylated product was apparently formed by tissue as follows: (a) cleavage of oleoyl-CoA by an amidase to form S-oleoylmercaptoethylamine; (b) spontaneous rearrangement to form N-oleoylmercaptoethylamide; and (c) enzymatic methylation of the free thiol by AdoMet. Participation of the amidase was suggested by the biosynthesis of the amide (free thiol) using [1-14C]oleoyl-CoA.

Acyl Coenzyme A↗

PubMed1981

Isoniazid metabolism in the rabbit, and the effect of rifampin pretreatment.

The metabolism of 14C-isonicotinyl hydrazide (INH) (50 mg/kg, po) was studied in male New Zealand White rabbits and the effect on INH metabolism of pretreating the rabbits for 7 days with rifampin (100 mg/kg po per day) was also studied. The 14C-labelled metabolites were separated and quantitated by TLC and the unlabelled hydrazino metabolites by GLC. Absorption and elimination of INH was rapid since the peak blood 14C level was attained by 1 hr and the T 1/2 of elimination was 2.67 +/- 0.36 hr. By 12 hr 68.5 +/- 4.1% of the dose was recovered in the urine. The major metabolite excreted in the urine was isonicotinic acid (INA) which accounted for 40.3 +/- 3.5% of the dose followed by acetylisoniazid (AcINH) at 15.8 +/- 1.2%. The relatively high proportion of INA excreted by the rabbit compared to the rat and human is attributed to a high level of amidase in the rabbit, and is suggested as a possible explanation for the rabbit's sensitivity to INH-induced hepatotoxicity. Rifampin pretreatment produced only one significant change in the parameters studied and that was a reduction in AcINH excreted in urine. It is suggested that this effect may be due to rifampin increasing hepatic amidase activity.

Animals↗

PubMed1994

Inhibitors of arachidonoyl ethanolamide hydrolysis.

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

Amidohydrolases↗

PubMedDate not supplied

[Asparaginase and glutaminase activity in Pseudomonas fluorescens in continuous cultivation].

The cells of Pseudomonas fluorescens AG contain two inducable asparaginase enzymes: one of them hydrolyzes only L-asparagine (asparaginase A), the other--L-asparagine, L-glutamine, and D-asparagine (asparaginase AG). In the conditions of continuous cultivation of the bacteria, aspartic and glutamic acids induce the formation of these enzymes only when the amino acids were used simultaneously as a growth-limiting factor and as a sole source of carbon and nitrogen. Both enzymes are not induced in the conditions when the growth is limited by the nitrogen of these amino acids. When the growth was limited by carbon, asparagine, aspartic and glutamic acids induce asparaginase AG more than asparaginase A. Asparagine and glutamine are better inductors than the corresponding amino acids. The activity of asparaginase and glutaminase increases with the specific growth rate of the culture. The induced synthesis of both amidases, after prolonged growth of the culture on a defined medium with glycerol, is inhibited by glycerol but not by glucose. The results are discussed from the viewpoint of regulation of amidases in these bacterial cells.

Asparaginase↗

PubMed1993

The biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked and O-linked glycans labeled by UDP-[6-3H]N-acetylgalactosamine.

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.

Animals↗

PubMed1993

Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum.

Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast. Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides. Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified. The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase. Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio. Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of [3H]Glc3[14C]Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic analysis of the products showed that [3H]Glc was removed only in the presence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor.

Carbohydrate Conformation↗

PubMed1998

[Inactivation and stabilization of urokinase fibrinolytic activity in vitro].

When incubated at 30 degrees C and pH 7.4, urokinase lost fibrinolytic activity (i.e., the plasminogen-activating activity measured by the time of fibrin clot lysis) but completely retained amidase activity. The enzyme inactivation rate depended on the urokinase concentration and, at concentrations of more than 1.5 microM, was described by a second order equation, which indicated that the enzyme underwent autolytic degradation (kaut = 3.8 x 10(-3) M-1 min-1). During incubation, urokinase (54 kDa) was converted into its low-molecular-mass form (33 kDa) and products of the A-chain degradation. The amidase activity did not correlate with the fibrinolytic activity in the cases when the enzyme molecule underwent local unfolding or partial degradation. The optimum mixture of agents for stabilizing the fibrinolytic activity of urokinase was found.

Albumins↗

PubMed2001

Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris.

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.

Base Sequence↗

PubMed1994

Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction.

A mechanism for bacteria to monitor the status of their vital cell wall peptidoglycan is suggested by the convergence of two phenomena: peptidoglycan recycling and beta-lactamase induction. ampG and ampD, genes essential for beta-lactamase regulation, are here shown to be required for recycling as well. Cells lacking either AmpG or AmpD lose up to 40% of their peptidoglycan per generation, whereas Escherichia coli normally suffers minimal losses and instead recycles 40 or 50% of the tripeptide, L-alanyl-D-glutamyl-meso-diaminopimelic acid, from its peptidoglycan each generation. The ampG mutant releases peptidoglycan-derived material into the medium. In contrast, the ampD mutant accumulates a novel cell wall muropeptide, 1,6-anhydro N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (anhMurNAc-tripeptide), in its cytoplasm. This work suggests that AmpG is the permease for a large muropeptide and AmpD is a novel cytosolic N-acetylmuramyl-L-alanine amidase that cleaves anhMurNAc-tripeptide to release tripeptide, which is then recycled. These results also suggest that the phenomenon of beta-lactamase induction is regulated by the level of muropeptide(s) in the cytoplasm, since an ampD mutation that results in beta-lactamase expression even in the absence of a beta-lactamase inducer coincides with accumulation of anhMurNAc-tripeptide. The transcriptional regulator AmpR is presumably converted into an activator for beta-lactamase production by sensing the higher level of muropeptide(s). This may be an example of a general mechanism for signaling the progress of external events such as cell wall maturation, cell division or cell wall damage.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2006

Increase of doxorubicin sensitivity for folate receptor positive cells when given as the prodrug N-(phenylacetyl) doxorubicin in combination with folate-conjugated PGA.

Folate receptor (FR) has been proposed as a promising target for tumor drug targeting. The aim of this study was to increase the chemo-sensitivity of FR-positive cells to doxorubicin by folate-directed enzyme prodrug therapy (FDEPT). Folate conjugated penicillin-G amidase was prepared and its ability to hydrolyze N-(phenylacetyl) doxorubicin was measured by HPLC. Fluorescence and confocal image analysis revealed that Folate-PGA can be specifically delivered into FR-positive HeLa and SKOV3 tumor cells. In vitro cytotoxity assays, IC50 was reduced with N-(phenylacetyl) doxorubicin versus doxorubicin for HeLa (3.1-fold reduction; p<0.001) and SKOV3 (3.3-fold reduction; p<0.001) when Folate-PGA was specifically bound to the cells. Complete activation was confirmed in HeLa and SKOV3 cells pretreated with free folic acid (1 mM), where the combination of N-(phenylacetyl) doxorubicin with Folate-PGA did not show any significant cell toxicity to the IC50 of doxorubicin. Pharmacokinetic clearance and biodistribution studies in vivo showed that 125I-Folate-PGA was cleared from blood within 24 h and had significantly higher tumor uptake compared to 125I-PGA (p<0.05). These results demonstrate that the FDEPT approach may be a potential promising strategy to improve chemotherapy-resistant cancers therapeutic ratio and warranted future studies.

Animals↗

PubMed1995

A method for monitoring the glycosylation of recombinant glycoproteins from conditioned medium, using fluorophore-assisted carbohydrate electrophoresis.

We have developed a method for monitoring the N-glycosylation of recombinant glycoproteins directly from conditioned medium samples. Proteins in the conditioned medium are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After staining the membranes with Coomassie blue, the protein(s) of interest is excised. Oligosaccharides are released from the membrane-bound glycoprotein by digesting with peptide N4-(acetyl-beta-glucosaminyl) asparagine amidase and labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS). Labeled oligosaccharides are then separated on polyacrylamide gels which allow for the direct comparison of samples. We have shown that recombinant human lysosomal hydrolase alpha-galactosidase A is N-glycosylated with both sialylated and phosphorylated oligosaccharides. ANTS-labeled oligosaccharide bands from alpha-galactosidase A were isolated from polyacrylamide gels. Sialylated and phosphorylated bands were identified by shifts in their electrophoretic mobility after digesting with neuraminidase or alkaline phosphatase to remove sialic acid or phosphate groups, respectively. Using the ANTS-labeled oligosaccharides from alpha-galactosidase A, we have shown that polyacrylamide gels can be used to resolve sialylated and phosphorylated oligosaccharide structures.

Amidohydrolases↗

PubMed2001

Fluorogenic assay for penicillin G acylase activity.

A simple, highly sensitive, and rapid assay for high-throughput screening of penicillin G acylase-producing bacteria is presented. The method is based on the specific release of fluorescent 7-amino-4-methyl-coumarin through cleavage of phenylacetyl-4-methyl-coumaryl-7-amide by penicillin G acylase. The present method is suitable for screening pure enzymes as well as various penicillin G acylases like those from Escherichia coli, Proteus rettgeri, and Kluyvera citrophila in cell extracts. In addition, the new substrate was used for rapid assay of amidase activity in nondenaturing polyacrylamide gels.

Coumarins↗

PubMed1995

Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth.

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.

Amino Acid Sequence↗

PubMed1998

Overexpression of PNGase at from baculovirus-infected insect cells.

Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme.

Amidohydrolases↗

PubMed1985

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli.

A 7.5 kb BclI-fragment of Streptococcus pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-L-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [35S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt+ phenotype.

Amidohydrolases↗