Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis

Collins, Mary Lynne Perille; Saltón, M. R. J.

doi:10.1016/0005-2736(79)90029-4

Cited by 54 publications

(16 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The extraction of M-2 from cells with GuHCI as described above suggested that GuHCI might also be used to dissociate M-2 from the peptidoglycan-enzyme complex. GuHCI (6 M) extracts of peptidoglycan-enzyme complex yielded about twice the M-2 activity yielded by alkaline extracts (pH 12) and 4 (4,10,25,26,33). We found that the chaotropic salts GuHCI, guanidine-thiocyanate, sodium thiocyanate, and lithium thiocyanate could be used to extract both muramidases from the intact bacterial cells and from the insoluble residue (crude cell wall fraction) of mechanically disrupted E. hirae.…”

Section: Resultsmentioning

confidence: 87%

“…Re-Nacetylation of the peptidoglycan did not significantly decrease the amount of peptidoglycan required to bind all of the M-2 activity in the supernatant culture medium (data not shown). The affinity of M-2 (and M-1) for its insoluble peptidoglycan or wall substrates appears to be very high; very high salt concentrations (23) or high pHs (4,17,18) are required to dissociate these complexes. The extraction of M-2 from cells with GuHCI as described above suggested that GuHCI might also be used to dissociate M-2 from the peptidoglycan-enzyme complex.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

View full text Add to dashboard Cite

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcusfaecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) was shown to possess two separate and distinct peptidoglycan hydrolase activities; both enzymes are N-acetylmuramoylhydrolases (muramidases) (18,29). Both enzymes are high-molecular-weight, complex proteins that possess a number of rather unusual properties, especially in contrast to avian and bacteriophage lysozymes that hydrolyze the same bond in the cell wall peptidoglycan. Muramidase-1 (M-1) was isolated and purified to homogeneity and was shown to occur in a latent, 130-kDa form that can be proteolytically hydrolyzed to an active, 87-kDa form (17,24,30). M-1 was also shown to be a glycoenzyme containing covalently attached monomeric and oligomeric glucose (17) and to possess approximately 12 phosphodiester-linked monomeric 5-mercaptouridine monophosphate residues (8). In addition, M-1 was shown to processively hydrolyze linear, soluble, un-cross-linked peptidoglycan chains (1).Muramidase-2 (M-2) was partially purified from the supernatant culture medium and was shown to be a 70-to 75-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (18). M-2 was also purified to apparent homogeneity from the insoluble pellet of disrupted E. hirae (7). Two polypeptide bands, one of about 125 kDa and the other of about 71 kDa, were show...

show abstract

Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…5). Triton X-100 is also a nonionic detergent, which extracts intrinsic and integral proteins from membrane components, solubilizes hydrophobic proteins, and forms micelles with them while maintaining their structure and activity (71,83,346). It can be used to release compartmentalized indicator.…”

Section: E Compartmentalizationmentioning

confidence: 99%

Measurement of Intracellular Calcium

Takahashi

Camacho

Lechleiter

et al. 1999

Physiological Reviews

667

511

View full text Add to dashboard Cite

To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

show abstract

“…This extract was partially purified by DEAE-cellulose chromatography, precipitation with (NH4)2SO4 at 45% saturation, and chromatography on Sephacryl 400 (Pharmacia Fine Chemicals, Piscataway, N.J.). New Zealand White female rabbits were immunized by subcutaneous injection with immunogen (5' pg of protein) in 50% Freund incomplete adjuvant (Difco) by the immunization protocol reported previously (10). The immunoglobulin fraction was purified from crude antiserum and concentrated by (NH4)2SO4 precipitation and dialysis (10).…”

mentioning

confidence: 99%

“…New Zealand White female rabbits were immunized by subcutaneous injection with immunogen (5' pg of protein) in 50% Freund incomplete adjuvant (Difco) by the immunization protocol reported previously (10). The immunoglobulin fraction was purified from crude antiserum and concentrated by (NH4)2SO4 precipitation and dialysis (10). To obtain antibody specific for components of phototrophic cells, this antibody preparation was adsorbed with membranes prepared from aerobically grown cells (20 mg of membrane protein per ml of purified antibody).…”

mentioning

confidence: 99%

Immunocytochemical ultrastructural analysis of chromatophore membrane formation in Rhodospirillum rubrum

Crook¹,

Treml²,

Collins³

1986

J Bacteriol

View full text Add to dashboard Cite

An immunocytochemical ultrastructural study of Rhodospirilum rubrum cultured under semiaerobic conditions was conducted to correlate the localization of functional components with membrane formation. R. rubrum is a facultatively phototrophic organism. Under reduced oxygen, this bacterium forms an intracytoplasmic chromatophore membrane that is the site of the photosynthetic apparatus. Immunogold techniques were used to localize intracellular protein antigens associated with the photosynthetic apparatus. Antibody, demonstrated by immunoblotting to be specific for the reaction center and light-harvesting photochemical components, was conjugated to colloidal gold particles and used for direct immunolabeling of fixed, sectioned specimens. Membrane invaginations appeared by 4 h after transition to induction conditions, and mature chromatophore membrane was abundant by 22 h. The occurrence of chromatophore membrane was correlated with bacteriochlorophyll a content and the density of the immunolabel. In uninduced (aerobic) cells and those obtained from cultures 0.5 h posttransition, the immunogold preferentially labeled the peripheral area of the cell. In contrast, in cells obtained after 22 h of induction, the central region of the cell was preferentially immunolabeled. These findings provided immunocytochemical evidence supporting the hypothesis that the chromatophore membrane is formed by invagination of the cytoplasmic membrane.Rhodospirillum rubrum is a facultatively photosynthetic bacterium that has been used as a model in studies of membrane structure and formation (22,36). Under aerobic conditions nonpigmented cells undergo chemotrophic metabolism. Under anaerobic conditions in the light, R. rubrum grows phototrophically. Bacteriochlorophyll a (BCHL) and carotenoids and associated proteins are localized in the intracytoplasmic chromatophore membrane. Ultrastructural and physical evidence has suggested that this chromatophore membrane is continuous with the cytoplasmic membrane (for a review, see reference 39). Moreover, studies revealing the opposite asymmetry of the chromatophore and cytoplasmic membranes in R. rubrum (34, 42) and other photosynthetic bacteria (8,11,25) imply the existence of a membrane continuum because the cytoplasmic face of the chromatophore membrane forms the outer surface of isolated chromatophores.The apparent continuity between the chromatophore and cytoplasmic membrane has led to the hypothesis that the chromatophore membrane is formed by invagination of the cytoplasmic membrane (35). Evidence to support this hypothesis is provided by the freeze-etching studies of Golecki and Oelze (13 Electron microscopy. Cells were harvested, washed, and suspended in a minimal amount of 10 mM potassium phosphate buffer (pH 7.0) and held at 0°C until cell blocks were prepared with 2% agarose (Seakem HGT; FMC Corp., Marine Colloids Div., Rockland, Maine). Double-distilled water used in all reagents was boiled for 10 min and filter sterilized. For cell block preparation, cell suspensions were broug...

show abstract

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis

Cited by 54 publications

References 50 publications

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Measurement of Intracellular Calcium

Immunocytochemical ultrastructural analysis of chromatophore membrane formation in Rhodospirillum rubrum

Contact Info

Product

Resources

About