Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.     

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.     

Immunoelectron microscopic localization of penicillinase in Bacillus licheniformis.

Penicillinase was localized in log-phase cells of Bacillus licheniformis 749/C by labeling with ferritin-anti-penicillinase immunoglobulin G conjugate. Mildly fixed homogenized cells, isolated subcellular fractions, and frozen thin sections were labeled. The label was distributed in discrete patches in the cell envelope. The patches extended from the inside part of the membrane to the outside part of the wall. The inside part of the membrane was labeled more extensively than the outside part. The cytoplasm also bound some ferritin-immunoglobulin G conjugate. Immunoelectrophoresis and biochemical assay of cytosol material suggest that the cytoplasmic antigenic sites are a protease-sensitive form of penicillinase.     

An immunoenzyme method of isolation of foot-and-mouth disease virus by using beta-lactamase conjugate with virus-specific antibodies

It was shown that in was feasible to use conjugates of virus-specific antibodies and beta-lactamase from Bacillus licheniformis 749/c to identify aphthosa virus antigens. The antigen titers determined by enzyme immunoassay (EIA) using a beta-lactamase conjugate were 5-64 times higher than the analogous indices of the complement fixation test. Unlike EIA, that by using the antibody conjugates with peroxidase or alkaline phosphatase there were observed no "background" responses.     

Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination.

When membranes of Bacillus licheniformis MC14 were extracted exhaustively with 1 M magnesium, approximately 80% of the membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], E.C. 3.1.3.1) was solubilized. The remaining activity could be extracted with a cationic detergent, hexadecylpyridinium chloride, without loss of enzymatic activity. The detergent-extractable alkaline phosphatase was immunoprecipitable with antibody to the salt-extractable alkaline phosphatase or the secreted alkaline phosphatase, had an approximate molecular weight of 60,000, and was localized 100% on the outer surface of the cytoplasmic membrane.     

Penicillinase-releasing protease of Bacillus licheniformis: purification and general properties.

The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein which differs from the exoenzyme in that it has an additional sequence of 24 amino acid residues and a phosphatidylserine at the NH2 terminus. In exponential-phase cultures, the conversion of membrane penicillinase to exoenzyme occurs at neutral and alkaline pH. An enzyme that will cleave the membrane penicillinase to yield the exoenzyme is present (in small amounts) in exponential-phase cells and is released during their conversion to protoplasts. The enzyme is found in the filtrate of a stationary-phase culture of the uninduced penicillinase-inducible strain 749 and has been purified to apparent homogeneity from this source. The protease has an approximate molecular weight of 21,500 and requires Ca2+ ions for stabilization. It has a pH optimum of 7.0 to 9.5 for hydrolysis of casein and for the cleavage of membrane penicillinase. Both activities are inhibited by diisopropylfluorophosphate; hence, the enzyme is a serine protease. This enzyme may be entirely responsible for the formation of exopenicillinase by this organism, since the other neutral and alkaline proteases of strain 749 have little, if any, activity in releasing exopenicillinase. The enzyme has been termed penicillinase-releasing protease.     

Interrelationship of carbohydrate metabolism and alkaline phosphatase synthesis in Bacillus licheniformis 749/c.

Membrane-bound alkaline phosphatase of Bacillus licheniformis 749/c is derepressed by glucose in complex and chemically defined media. In the presence of lactate, pyruvate, or succinate the synthesis is repressed. The lactate repression neither affects total protein synthesis nor inhibits penicillinase synthesis. Thus, carbon sources specifically influence alkaline phosphatase synthesis. Although variations in the inorganic phosphate content of the growth media directly affect alkaline phosphatase synthesis, the intracellular inorganic and total phosphate pools appear to be unrelated to its repression or derepression. During lactate repression there is preferential incorporation of lactate molecules into glycogen, whereas no such incorporation could be detected from glucose. Net glycogen synthesis remains the same in glucose- or lactate-grown cells. It is postulated that, in phosphate-deficient growth medium, gluconeogenic metabolism regulates alkaline phosphatase synthesis.     

Distribution in clusters of complement receptor type one (CR1) on human erythrocytes

The distribution of CR1 on human E was studied using label-fracture and thin section electron microscopy. CR1 was found to be organized in clusters on unfixed cells and on cells that had been prefixed with paraformaldehyde or glutaraldehyde before labeling. The number of clusters/E ranged from 8 to 20 as estimated from the examination of freeze-fracture replicas of labeled cells. Clusters contained an average of 30 to 75 gold particles on cells from two donors which expressed 462 and 586 CR1 Ag sites/cell, as determined by flow cytometry. In thin section electron micrographs, gold complexes were seen surrounding an electron-dense material protruding from the membrane which represents compact aggregates of CR1. The maximal distance between gold particles and the membrane was 100 nm, which corresponds to the estimated length of the major allotypic form of CR1, as calculated from the primary DNA sequence of the molecule. The distribution in clusters of CR1 on the E membrane may provide the basis for an enhanced affinity of C3b-CR1 interactions on the plasma membrane of the cells and may explain the preferential binding of C3b-bearing immune complexes to E in vivo.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Vectorial and nonvectorial transphosphorylation catalyzed by enzymes II of the bacterial phosphotransferase system

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.     

Characteristics of Secretion of Penicillinase, Alkaline Phosphatase, and Nuclease by Bacillus Species

The distribution of alkaline phosphatase and nuclease activity between cells and medium was examined in one strain of Bacillus licheniformis and four strains of B. subtilis. Over 95% of both activities was found in the medium of the B. licheniformis culture, but in the B. subtilis cultures the amount of enzyme activity found in the medium varied with the strain and the enzyme considered. B. licheniformis 749 and its penicillinase magnoconstitutive mutant 749/C were grown in continuous culture with phosphorous as the growth-limiting factor, and the kinetics of penicillinase formation and secretion were examined. Nutrient arrest halted secretion (usually after a lag of about 30 min) in both the inducible and constitutive strains. Chloramphenicol did not eliminate secretion, but under certain circumstances reduced its rate. In the inducible strain treated with a low level of inducer, the rate of secretion was more affected by the rate of synthesis than by the level of cell-bound enzyme. During induction, the onset of accretion of cell-bound penicillinase and secretion of the exoenzyme were nearly simultaneous. It seems unlikely that a long-lived, membrane- or cell-bound intermediate is mandatory in the secretion of the three enzymes by Bacillus species. In the case of penicillinase secretion, there are at least two different phases. When penicillinase synthesis is proceeding rapidly, the rate of secretion is five to six times greater at equivalent concentrations of membrane-bound penicillinase than it is when penicillinase synthesis is reduced. The data require that any membrane-bound intermediate in the formation of exoenzyme be much shorter-lived in cells with a high rate of synthesis than in cells with a low rate. Either there are two separate routes for the secretion of penicillinase or the characteristics of the process vary substantially between the early stages and the declining phase of induction.     

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Cloning and characterization of the Bacillus licheniformis gene coding for alkaline phosphatase.

The structural gene for alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) of Bacillus licheniformis MC14 was cloned into the Pst1 site of pMK2004 from chromosomal DNA. The gene was cloned on an 8.5-kilobase DNA fragment. A restriction map was developed, and the gene was subcloned on a 4.2-kilobase DNA fragment. The minimum coding region of the gene was localized to a 1.3-kilobase region. Western blot analysis was used to show that the gene coded for a 60,000-molecular-weight protein which cross-reacts with anti-alkaline phosphatase prepared against the salt-extractable membrane alkaline phosphatase of B. licheniformis MC14 .     

Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], EC 3.1.3.1) expressed in two human osteosarcoma cell lines (Saos-2 and KTOO5) in culture was the tissue nonspecific type and was released from the plasma membrane by phosphatidylinositol (PI) phospholipase C. Despite a difference of 10-fold between the two cell lines in the amount of alkaline phosphatase expressed, the phospholipase solubilized nearly all of the phosphatase from resuspended cells of the two lines. Alkaline phosphatase released with Nonidet-P40 from Saos-2 cells had a Mr of 445,000 by gradient gel electrophoresis in the absence of detergent; that released by PI-phospholipase C was 200,000. The subunit Mr of both solubilized forms was 86,000. Thus, tetrameric alkaline phosphatase in the membrane is attached by a PI-glycan moiety and is converted to dimers when released by PI-phospholipase C. Tunicamycin treatment of Saos-2 cells in culture affected the release of alkaline phosphatase by a high concentration of PI-phospholipase C, but not by a low concentration; both the rate and extent of release were lower from treated cells. However, the enzyme released from the treated cells was in two forms with different molecular weights; it seems that both glycosylated and nonglycosylated dimers were transported to the cell surface and incorporated into the plasma membrane. Glycosylation does not appear to be necessary for alkaline phosphatase to be anchored in the membrane via PI.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

Localization of Cell-bound Penicillinase in Bacillus licheniformis

When protoplasts are prepared from Bacillus licheniformis (strain 749/C, constitutive for penicillinase), approximately 60% of the cell-bound penicillinase is released. The remainder is retained by the protoplast and cannot be removed by washing. This release is specific, in that less than 7% of the cellular reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and alpha-glucosidase is liberated by the treatment. The freed penicillinase is excluded from G-200 Sephadex, and it is partially sedimented with a force of 65,000 x g for 20 hr. It is probably attached to characteristic tubular and vesicular structures with single-layered membranes that are comparable to structures previously described in intact penicillinase-forming cells. The specific activity of the organelle is more than six times that of twice washed peripheral membrane; furthermore, about 8% of the protein of the structure is penicillinase. At substrate concentrations (benzylpenicillin) of about one-fifth the K(m) value, whole cells show a slight permeability restriction, although this does not occur in isolated particles and protoplasts.     

The ultrastructural localization of adenosinetriphosphatase and alkaline phosphatase activity in eosinophil leukocytes

Summary The previously undescribed localization of reaction products of adenosinetriphosphatase and of alkaline phosphatase in eosinophil leukocytes was demonstrated by cytochemical studies of the rat intestine. Alkaline phosphatase reaction product was found only in minimal amounts on the plasma membrane but was distinct on the nuclear membranes and outer compartment of mitochondria but not on the cristae. The Golgi membranes and the endoplasmic reticulum reacted but less intensely. The specific granules showed no alkaline phosphatase activity.The adenosinetriphosphatase reaction, on the other hand, was found on the plasma membrane, vesicular or tubular profiles of the endoplasmic reticulum and on the matrix of the specific granules. The crystalloid of the granules did not show any reaction.Recipient of a postdoctoral fellowship from the muscular distrophy association of Canada.