Isolation and Characterisation of Glycoproteins from Sputum

Roberts, G P

doi:10.1111/j.1432-1033.1974.tb03895.x

Cited by 83 publications

(39 citation statements)

References 52 publications

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“…,y-Aminobutyric acid (GABA) is widely believed to be the most important chemical mediator of inhibition in the vertebrate central nervous system. The specificity of the inhibitory postsynaptic responses elicited by GABA has suggested that in postsynaptic membranes of GABA-ergic synapses there is a well-defined recognition site for GABA that is coupled with the Cl-ionophore (1). Indeed, binding assay studies using radioactive GABA, crude synaptic membranes, and a Na+-free buffer have shown that GABA recognition sites with characteristics consistent with those expected at postsynaptic sites of GABA-ergic synapses, are present in the mammalian brain (2)(3)(4)(5).…”

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confidence: 99%

Purification of an endogenous protein inhibitor of the high affinity binding of gamma-aminobutyric acid to synaptic membranes of rat brain.

Toffano¹,

Guidotti²,

Costa³

1978

Proc. Natl. Acad. Sci. U.S.A.

148

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In a medium without Na+, y-aminobutyric acid jGABA) binds at 00 to freshly prepared crude synaptic memranes from rat cerebral cortex with an apparent dissociation constant of 218 nM. An endogenous inhibitor of the Na+-independent GABA binding was removed from these membranes freezing and thawing and by repeated washing with Tris citrate buffer (50 mM, pH 7.1) containing 0.01% Triton X-100. As a result, the crude synaptic membranes bind GABA at 00 with two dissociation constants, 20 nM and 111 nM. The endogenous inhibitor is a thermostable (950 for 15 min) acidic protein of approximately 1.5 X 104 daltons. It was purified (about 500-fold) with a series of procedures including gel chromatography on Sephadex G-100 and ion exchange chromatography on Dowex 50W-X8 (Hf). Recombination of the purified endogenous inhibitor with crude synaptic membrane preparations deprived of the endogenous inhibitor showed that the purified inhbitor blocked noncompetitively the sites for hi-affinity GABA binding. A role of this endogenous regulator in the function of GABA-ergic synapses is discussed. ,y-Aminobutyric acid (GABA) is widely believed to be the most important chemical mediator of inhibition in the vertebrate central nervous system. The specificity of the inhibitory postsynaptic responses elicited by GABA has suggested that in postsynaptic membranes of GABA-ergic synapses there is a well-defined recognition site for GABA that is coupled with the Cl-ionophore (1). Indeed, binding assay studies using radioactive GABA, crude synaptic membranes, and a Na+-free buffer have shown that GABA recognition sites with characteristics consistent with those expected at postsynaptic sites of GABA-ergic synapses, are present in the mammalian brain (2-5). In addition to the postsynaptic recognition sites, brain synaptosomes contain GABA recognition sites associated with GABA uptake (for a review, see ref. 6); however, these sites can be differentiated from the GABA postsynaptic recognition sites because they are Na+-dependent (2-5). The KD for the Na+-independent GABA binding to freshly prepared crude synaptic membranes from rat brain is higher than that for other putative neurotransmitters such as enkephalins (7), dopamine (8), and epinephrine or norepinephrine (9). The freshly prepared crude synaptic membranes require several freeze-thawing steps and subsequent washes and treatment with Triton X-100 in order to lower by about 1:10 the KD value for Na+-independent GABA binding. This value is similar to that for postsynaptic recognition sites of other natural ligands (5, 7-9).Usually treatment of the crude synaptic membranes with Triton X-100 either denaturates or solubilizes neurotransmitter receptors bound to these membranes; however GABA binding sites appear to be unique in that they remain in the membrane after detergent treatment and show enhanced binding affinity for the endogenous ligand (5). It has been suggested that these washings and treatment with Triton X-100 are required in orderThe costs of publication of this article ...

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confidence: 99%

Purification of an endogenous protein inhibitor of the high affinity binding of gamma-aminobutyric acid to synaptic membranes of rat brain.

Toffano¹,

Guidotti²,

Costa³

1978

Proc. Natl. Acad. Sci. U.S.A.

148

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“…It resembles mucins purified by others from several human and animal organs ( I , 4, 6, [17][18][19]. in that it is a polydisperse viscous glycoprotein rich in carbohydrate.…”

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Effects of Calcium on Intestinal Mucin: Implications for Cystic Fibrosis

Forstner

1976

Pediatr Res

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“…Since proteinases in the C F LM-gel samples fragmented radiolabeled tracheobronchial mucin In vitro (Fig. 4), proteinases in LM-gel samples from pathological airways may account for the variations in molecular weight, solubility, and chromatographic behavior reported for tracheobronchial mucins (6,(37)(38)(39)(40)(41)(42)(43)46). This study has also demonstrated that TBM from healthy individuals exhibited physical and chemical properties similar to those of TBM from a patient with bronchial asthma (5).…”

Section: Discussionmentioning

confidence: 59%

“…4) The cysteine contents of the CF lung mucins analyzed were considerably lower than those of normal and asthmatic mucins, and similar to values obtained on analysis of the proteolytically-digested glycopeptide fragments of asthmatic mucin (39,40). However, considerable variability in the cysteine concentrations of tracheobronchial mucins has been reported (5,6,33,(38)(39)(40)(41)(42)(43) and cysteine is often not measured quantitively as it requires almost a mg of purified mucin for accurate measurements in duplicate.…”

Section: Discussionmentioning

confidence: 98%

Biochemical Properties of Tracheobronchial Mucins from Cystic Fibrosis and Non-Cystic Fibrosis Individuals

et al. 1987

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ABSTRACT. Tracheobronchial mucins from healthy individuals and from patients with bronchial asthma or cystic fibrosis (CF) were isolated from lung mucus, purified, and their chemical and physical properties compared. Normal and asthmatic mucins required both a dissociating and a reducing agent for solubilization and exhibited identical chromatographic behavior on Sepharose 4B, Sepharose 2B, and hydroxylapatite and similar amino acid and carbohydrate compositions. In contrast, 1) C F lung mucins were solubilized in the absence of dissociating and/or reducing agents and 2) the majority of the C F mucins analyzed was eluted in the included volume of Sepharose 4B with K. , values of 0.3 + 0.1 rather than in the void volume and thus appeared smaller than normal and asthmatic mucins. The lower molecular weight mucins in C F sputum apparently are produced by bacterial or inflammatory cell proteinases since radiolabelled asthmatic mucin was digested to smaller fragments when incubated with crude C F lung mucosal samples. Furthermore, mucins secreted by tracheal explants from C F and from non-CF individuals eluted in the void volume on Sepharose 4B, suggesting that CF tracheobronchial mucins were not inherently smaller than non-CF mucins. (Pediatr Res 22: 545-551, 1987 mucus of exocrine glands or epithelial tubular systems of the respiratory, gastrointestinal, and reproductive tracts (2) which is the basis for the generally held assumption that C F mucus is "abnormal." Since mucus glycoproteins (mucins) are the macromolecules responsible for the viscoelastic properties of mucus (3-5), alterations in mucin structure could effect the physiological behavior of mucus.To date, the most comprehensive studies on C F mucins (6-8) have utilized TBM isolated from C F sputum as copious amounts of such material are readily available. There is limited information available on TBM from healthy airways primarily because of the difficulties in obtaining sufficient amounts of adequate material for analysis. Thus, insufficient information currently exists to determine whether C F and non-CF mucins are different (see Ref. 9 for review) although available studies suggest differences in sulfation of C F respiratory tract mucins (10). However, recent studies have suggested that TBM from healthy airways exhibit physical properties (size, chromatographic behavior on molecular sieving, solubility) different than those of mucins from pathological airways (I I-13). While isolating and purifying human mucins from normal and pathological mucus, we obtained results that indicated that C F TBM were considerably more heterogeneous and smaller in size than non-CF TBM. The smaller size of C F mucins in the samples analyzed could result from in vivo proteolysis, as C F airways are chronically infected with pathogens that produce proteinases (14, 15) and both C F sputum and bronchial washings contain high levels of extracellular proteinase activity (1 6-18). Alternatively, the smaller mucins could reflect inherent differences in C F mucins. These possibili...

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Isolation and Characterisation of Glycoproteins from Sputum

Cited by 83 publications

References 52 publications

Purification of an endogenous protein inhibitor of the high affinity binding of gamma-aminobutyric acid to synaptic membranes of rat brain.

Purification of an endogenous protein inhibitor of the high affinity binding of gamma-aminobutyric acid to synaptic membranes of rat brain.

Effects of Calcium on Intestinal Mucin: Implications for Cystic Fibrosis

Biochemical Properties of Tracheobronchial Mucins from Cystic Fibrosis and Non-Cystic Fibrosis Individuals

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