A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides

Ludowieg, Julio; Dorfman, Albert

doi:10.1016/0006-3002(60)91233-6

Cited by 180 publications

(33 citation statements)

References 20 publications

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“…Hadzija's assay (17) was used for muramic acid, with pure muramic acid as the standard (Sigma; reagent grade); the total hexosamine assay was that described by Ghuysen et al (11). Hexosamine N-acetyl groups were estimated by Ludowieg's method (55).…”

Section: Methodsmentioning

confidence: 99%

Elasticity of the sacculus of Escherichia coli

1992

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Preparations of purified peptidoglycan ofEscherichia coli (i.e., sacculi) were studied by low-angle laser light scattering. Control experiments and theoretical calculations based on the Rayleigh-Gans theory showed that the mean sacculus surface area could be accurately inferred from measurements with our apparatus by using computer routines developed previously. Large changes in the mean saccular surface area resulted from alterations in the stress caused by varying the net charge on the sacculi. The net charge was affected by altering the suspending medium pH, causing carboxyl and amino groups in the peptidoglycan to gain or lose protons, or by acetylation or succinylation of the amino groups. A preponderance of either plus or minus charges caused an expansion of the mean sacculus surface area. The largest increase in area probably represents the elastic limit of the peptidoglycan and was 300%o above the area of isoionic sacculi. This degree of expansion is consistent with possible conformations of the intact peptidoglycan structure without necessitating rupture of the wall fabric. Our findings concerning saccular elasticity provide support for the surface stress theory. It provides a mechanism so that bacteria can grow and divide while maintaining turgor pressure, without the necessity of having and using proteins to do the mechanical work.It is now well established that the peptidoglycan sacculus is the stress-resistant, shape-determining structure covering the procaryotic cell (4, 23, 27-29, 34, 37, 42, 45, 46, 49, 63, 72). Its covalent structure makes the sacculus the largest macromolecule in nature and imparts strength to the bacterial wall. Additionally, the ability of the sacculus to stretch is of fundamental importance, both for giving flexibility to the organism and providing a mechanism for its enlargement during growth. The elasticity, as measured by Young's modulus, is the inverse of the ratio that a body is stretched (strain) by a given force (stress) (71). At high-enough stress, a body's elastic limit is reached, and it cannot be stretched further without the rupture of integral covalent bonds. Elastic enlargement of a single cell's wall fabric, composed of cross-linked peptidoglycan, depends on conformational changes that involve only rotations and easily made rearrangements of hydrogen bonds. Enlargement exceeding the elasticity must involve the rupture (hydrolysis in an aqueous environment) of covalent bonds (37,40).Sacculi are too small to have either their elasticity or their rupture limit (extensibility) measured by engineering materials testing methods (71). Although the sacculus cannot be affixed at two ends and mechanically stretched, the relationship between the sacculus net charge and surface area can be determined. We studied sacculi of Escherichia coli from cultures in balanced exponential growth. They were prepared free of almost all nonpeptidoglycan materials. The mean surface area of the sacculi under different conditions was measured by a low-angle laser-light-scattering method. ...

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Section: Methodsmentioning

confidence: 99%

Elasticity of the sacculus of Escherichia coli

1992

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“…Acetyl groups were determined by the method of Ludowieg and Dorfman [6]. Total phosphate was measured according to Chen et al [7] after the samples had been digested with perchloric acid as given by Shaw [8].…”

Section: Methodsmentioning

confidence: 99%

Isolation and Study of the Composition of a Peptidoglycan Complex Excreted by the Biotin‐Requiring Mutant of Brevibacterium divaricatum NRRL‐2311 in the Presence of Penicillin

Keglević

Ladešić

Hadžija

et al. 1974

European Journal of Biochemistry

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When cultures of biotin-requiring mutant of Brevibacterium divaricutum NRRL-2311 were treated in early logarithmic phase with penicillin (2 -3 units/ml) in a glucose-mineral medium under excess supply of biotin, large amounts of a high-molecular-weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of I-h and 24-h penicillin-treated cultures, showed that the main constituents were peptidoglycan components of noncross-linked structure bearing both L-and D-alanine residues ; evidence was also obtained for the occurrence of extractable lipid material, non-amino sugars and organic phosphate.Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin-requiring mutant of M . glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricutum strain. This suggests that excretion of peptidoglycan material by the two biotin-requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross-link formation in peptidoglycan induced by penicillin.Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin-treated Brev. mutant. Suitable labelling experiments with L-[U-14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion.During our studies on the accumulation of amino acids into the culture broth of the biotin-requiring mutant of Brevibacteriurn divaricatum (NRRL-231 l ) , it has been observed that in the presence of low doses of penicillin the extracellular accumulation of glutamate is accompanied by the excretion of some highmolecular-weight material. The first observations were made in cultures grown on glucose media containing biotin, to which penicillin G (2-10 units/ml) was added at the early logarithmic phase; incubations were carried out at 28 "C for 24, 48 and 72 h, respectively.When a growing culture was incubated simultaneously with 14C-labelled L-glutamic acid and penicillin for 24 h and the supernatant fluid submitted to gel fractionation, a substantial portion of radioactivity eluted a t low retention volumes. I n contrast, in the culture fluid of the control from which penicillin had been omitted, no radioactivity associated with some high-molecular-weight material could be detected. When subjected to paper electrophoresis and chromatography, the radioactive material excreted by penicillin-treated cells did not move from the origin. Hydrolysis of a sample in 6 M HCl a t 100 "C revealed diaminopimelic acid as one of the amino acid components.Since diaminopimelate is present in no other cellular material than in murein, the excreted mater...

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“…Nitrogen was determined by a micro-Kjeldahl procedure. 'Acetyl' groups were determined by the procedure o f L u d o w ieg and D o ri man (15); maximum values were obtained after methanolysis for 2 h.…”

Section: Methodsmentioning

confidence: 99%

Glomerular Basement Membrane in Experimental Nephrosis: Chemical Composition

Misra¹,

Kalant²

1966

Nephron

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Basement membrane prepared by ultrasonic vibration of isolated glomeruli was obtained from normal rats and from rats with anti-kidney serum nephrosis. The membrane was found to contain collagen, lipids, and at least two glycopeptides which were separable by chromatography on Seρhadex-G75 after removal of collagen by collagenase. Sialic acid was associated with collagen and with one of the glycopeptides. Acid hydrolysis released all the sialic acid in a dialyzable form. Neuraminidase released the same amount of sialic acid but only half was dialyzable, indicating that the remainder was not present in a terminal position. Nephrotic membrane contained less phospholipid and acetyl than normal, but the same amount of collagen, hexosamine and sialic acid. The sialic acid was liberated more readily by chemical hydrolysis, but less readily by neuraminidase than that of normal membrane; the collagen was also more readily degraded. It is concluded that in nephrosis, the amount of basement membrane material is not increased and that the membrane thickening seen microscopically, and the increased permeability, result from a change in organization at a molecular level.

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A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides

Cited by 180 publications

References 20 publications

Elasticity of the sacculus of Escherichia coli

Elasticity of the sacculus of Escherichia coli

Isolation and Study of the Composition of a Peptidoglycan Complex Excreted by the Biotin‐Requiring Mutant of Brevibacterium divaricatum NRRL‐2311 in the Presence of Penicillin

Glomerular Basement Membrane in Experimental Nephrosis: Chemical Composition

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