New centrifugation technique for isolating enzymes from large cell structures: isolation and characterization of two Bacillus subtilis autolysins

Bacillus subtilis cell walls can be centrifuged through a linear gradient of 0 to 2 m LiCl and 10 to 25% sucrose so that different autolysins are removed by different salt concentrations and banded in separate positions as the walls pass through the gradient. Using this technique we have found that B. subtilis cell walls are isolated with two autolytic enzymes attached. One autolysin, a glycosidase, can be eluted from walls with 0.5 m LiCl, has a pH optimum between 5 and 8, is relatively heat-sensitive, and has a molecular weight of 60,000. The other autolysin, an alanine amidase, can be eluted from walls with 1.5 m LiCl, has a pH optimum around 8, is relatively heat-stable, has a molecular weight of 35,000, and is present in quantities ten times greater than the glycosidase.     

Amidase activity involved in peptidoglycan biosynthesis in membranes of Micrococcus luteus (sodonensis).

Membrane suspensions prepared from Micrococcus luteus (sodonensis) in both the exponential and stationary phases of growth contained a transglycosidase activity capable of synthesizing linear peptidoglycan. Exponential-phase membranes also contained an N-acetylmuramyl-L-alanine amidase activity which degraded the peptidoglycan as it was formed. The product of this amidase was purified and found to be free pentapeptide. The amidase was specific for peptidoglycan and could not attack lower-molecular-weight substrates even though the susceptible bond was present. Crude cell wall preparations isolated from exponential-phase cells also contained high levels of amidase. This cell wall-bound amidase would preferentially degrade in vitro-synthesized peptidoglycan over its own cell wall. Amidase activity could be solubilized from both cell walls and membranes by Triton X-100 treatment, butanol extraction, or LiCl extraction. Both membrane- and cell wall-derived amidases, solubilized by LiCl extraction, appeared to be of high molecular weight (greater than 150,000). Once solubilized, these wall- and membrane-derived amidases could attack the cross-bridged peptidoglycan of purified native cell walls, whereas bound amidases could not.     

Micrococcus lysodeikticus bacterial walls as a substrate specific for the autolytic glycosidase of Bacillus subtilis

The Bacillus subtilis 168 autolytic glycosidase degrades Micrococcus lysodeikticus cells or cell walls, whereas the B. subtilis autolytic amidase does not. The criteria used to establish this fact included: the determination of chemical bonds broken, heat-inactivated kinetics, pH dependence curves, and the physical separation of glycosidase from amidase. The physical separation involved LiCl elution from two different ion-exchange materials, walls from B. subtilis 168 strain βAO, and walls from mutant strain βA173 derived from strain βAO. No evidence was obtained for B. subtilis vegetative bacteria making any more autolysins than one autolytic amidase and one autolytic glycosidase.     

Substrate specificity and some physicochemical properties of autolytic enzymes of the bacterium <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL 1

The substrate specificity of autolytic enzymes of the bacterium Lysobacter sp. XL 1 has been established. The periplasmic enzyme A8, the cytosolic enzyme A1, and the enzyme A10 solubilized from the cell walls and membranes with Triton X-100 exhibit glucosaminidase activity; the cytosolic enzyme A4 and the enzyme A9 solubilized from the cell walls and membranes with LiCl exhibit the muramidase activity. The cytosolic enzymes A3 and A6 have N-acetylmuramoyl-L-alanine amidase activity, and the enzyme A5 exhibits the diaminopimelinoyl-alanine endopeptidase activity. Some physicochemical properties of the most active autolytic cytosolic enzymes of Lysobacter sp. XL 1 (endopeptidases A5 and A7 and N-acetylmuramoyl-L-alanine amidase A6) were studied. The enzymes exhibit maximal activity over a wide range of buffer concentrations in weakly alkaline medium and moderate temperatures. The investigated enzymes are comparatively thermolabile proteins.     

Effect of dithiothreitol on activity and protein structure of human urine urokinase

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.     

Interaction of the pneumococcal amidase with lipoteichoic acid and choline

The choline-containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N-acetyl-muramoyl-L-alanine-amidase (amidase) in aqueous solution [H?ltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690-1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA-Sepharose. The amidase could be eluted from the column with choline only. This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA-Sepharose, the amidase converted from the applied E-form (an inactive form of the amidase) to the active C-form, a process which up to now was known to be mediated only by the pneumococcal choline-containing wall teichoic acid. Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from choline-grown cells: the membrane-associated enzyme was present in the C-form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase activity in membrane fractions derived from ethanolamine-grown pneumococci, in which choline containing LTA is absent. The LTA-Sepharose-associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase-digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA. When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase-deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wall-mediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C-amidase was different: the cell-wall-converted enzyme was of high Mr, whereas the choline-converted and the LTA-Sepharose-eluted enzyme showed an apparent low molecular mass known for the E-form, when analyzed on sucrose gradients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the N-acetylmuramic acid L-alanine amidase from Bacillus subtilis.

The N-acetylmuramic acid L-alanine amidase from Bacillus subtilis W-23 has been purified to apparent homogeneity. The enzyme is a monomer of molecular weight 51,000, which binds extremely tightly to homologous cell walls but not to heterologous cell walls, even of the closely related strain B. subtilis ATCC 6051. This difference in binding is only in part due to differences in teichoic acid between these two strains and to a large extent appears to represent differences in the arrangement of the peptidoglycan. A comparison of the amidase from B. subtilis W-23 and the enzyme previously purified from B. subtilis ATCC 6051 (Herbold and Glaser, 1975) shows that the two proteins, which cleave the same bond and are of the same size, do not cross-react immunologically and that the two enzymes are, therefore, not closely related in structure.     

Electron spin resonance study of the role of nitrosyl-heme in the activation of guanylate cyclase by nitrosoguanidine and related agonists.

One major component of lens plasma membrane is a glycoprotein that SDS-polyacrylamide gel electrophoresis shows to possess an apparent molecular weight of 26,000. When this protein is solubilized in low ionic strength buffers containing SDS, and heated to 100° for 1 to 3 min prior to electrophoresis, conversion into high molecular weight aggregate results. The heat lability of this protein is greatly enhanced if it solubilized and heated in buffers containing 0.1 M NaCl. At this ionic strength, incubation for 3 h at 38° results in conversion of 20% of the protein into high melecular weight aggregates. Most other membrane proteins isolated from lens membrane are insensitive to heat treatment. It is concluded that temperature and ionic strength must be recorded and controlled carefully when using SDS-polyacrylamide gel electrophoresis to study this membrane protein.     

Purification of human active urinary kallikrein: comparative inhibition studies of kininogenase and amidolytic activities

Large scale purification of human active urinary kallikrein is described. The final preparation was found homogeneous by means of SDS Page electrophoresis, amino acid composition and N-terminal analysis. The apparent molecular weight, determined on SDS Page electrophoresis, was 4.4 X 10(4). Comparative inhibition studies of the kininogenase and the amidase activities pointed out differences in the sensitivity of these two activities. Sodium inhibited amidase activity whereas kininogenase activity required the presence of this cation. In contrast, kininogenase activity was more sensitive to cadmium inhibition than amidase activity. Antibody against purified kallikrein did not completely inhibit amidase activity in crude urine. These discrepancies are consistent with the existence of several amidase activities in urine and also with possibly distinct catalytic sites on the same molecule, accordingly consideration of the methodology used appears very important when comparing results from different studies.     

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis.

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.     

Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.     

Intracellular location of the autolytic N-acetylmuramyl-L-alanine amidase in Bacillus subtilis 168 and in an autolysis-deficient mutant by immunoelectron microscopy.

Antisera against purified autolytic N-acetylmuramyl-L-alanine amidase from Bacillus subtilis 168 were prepared in rabbits. They neutralized the enzymatic action of the purified amidase acting on isolated sodium dodecyl sulfate (SDS)-treated walls from the same organism. They also inhibited the lysis of native walls, but only after the walls lysed partially. Amidase adsorbed to insoluble walls still combined with antibody. Antisera did not stop the lysis of whole cells. Lowicryl HM20 sections of both strain 168 and its autolytic mutant strain FJ6 were prepared by the progressive-lowering-of-temperature technique, immunolabeled with the antisera, and visualized with colloidal gold particles as markers. The highest concentration of gold particles seemed to be in the septa of dividing cells, followed by the side walls. There was some labeling of the cytoplasm. Adsorption of sera with SDS-treated walls reduced the overall labeling of sections considerably but did not alter the relative intracellular distribution of particles. The results for strains 168 and FJ6 were similar. Labeling of SDS-treated walls unexpectedly revealed the presence of a wall-bound amidase fraction.     

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.     

Purification and Properties of beta-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced beta-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher beta-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had beta-N-acetylgalactosaminidase activity equivalent to about 40% of its beta-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for beta-N-acetylglucosaminidase activity and 5.5 to 6.5 for beta-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

Molecular Weight of Human Brain Neutral Sphingomyelinase Determined In Situ by the Radiation Inactivation Method

The radiation inactivation method was used to determine the molecular weight of membrane-bound neutral sphingomyelinase from normal human brain. Inactivation curves showed a molecular mass of 167,000 +/- 32,000. Molecular weights of two control enzymes, beta-N-acetylglucosaminidase and nonspecific beta-glucosidase, determined by the same procedure, were consistent with previous reports.     

Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.     

N-acetylmuramyl-L-alanine amidase of Bacillus licheniformis and its L-form

A cell wall lytic enzyme has been demonstrated to be a component of the membrane of Bacillus licheniformis NCTC 6346 and an l-form derived from it. The lytic enzyme, characterized as an N-acetylmuramyl-l-alanine amidase, is solubilized from membranes by nonionic detergents. Ionic detergents inactivate the enzyme. In the bacterium the specific activities of amidase and d-alanine carboxypeptidase in mesosomes are approximately 65% of those in membranes. Selective transfer of lytic enzyme from nongrowing L-forms, L-form membranes, and protoplasts to added walls occurred after mixing, and 31 to 77% of the enzyme lost from L-form membranes was recovered on the walls. Membranes isolated from L-forms growing in the presence of added walls contained as little as 13% of the amidase found in membranes of a control culture. These results have been interpreted as showing that in vivo the amidase is "bound" to the surface of the bacterial cell membrane in such a location that it can be readily accessible to the cell wall.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.