Lysozyme: Ein Kapitel Molekularbiologie

Jollès, Pierre

doi:10.1002/ange.19690810703

Cited by 19 publications

(4 citation statements)

References 110 publications

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“…( with what was reported for others, e.g. lysozyme [33], a-amylase (which also has buried thiol groups) [34]. I n addition, the lack of free thiol groups agrees with the insensitivity of sucrase activity towards p-chloromercuribenzoate 121.…”

Section: 98supporting

confidence: 82%

A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition

Cogoli

Mosimann

Vock

et al. 1972

European Journal of Biochemistry

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A simplified procedure for the isolation of the sucrase -isomaltase complex from rabbit small intestine was worked out, which allows the preparation of this protein in a homogeneous form in good yield.The sucrase * isomaltase complex from rabbit small intestine is a glycoprotein. It is rich in acidic amino acids; it has no free thiols in the native state but denaturation unmasks six thiols. The tyrosine/tryptophan ratio is 2. The sugar moiety(ies) (15O/,) is composed of D-glucose, D-galactose, D-mannose, fucose, glucosamine and galactosamine (the latter two are probably acetylated). The sucrase isomaltase complex contains no neuraminic, uronic acid, lipids, phosphate, sulfate or zinc.The partial specific volume, calculated from the amino acid and sugar composition, agrees with that experimentally determined. The hydrophobicity coefficient of this membrane protein does not differ from that of other membrane bound or water soluble proteins.The sucrase isomaltase complex is associated with, or a building block of, the membrane of smallintestinal brush borders (for a review see [l]). It is one of the relatively few membrane proteins which can be solubilized and isolated in good yield [2,3]. The major reason for our interest in this oligo-enzyme complex is its close relationship with the transport of some sugars across the brush border membrane [4-6] and the demonstration of its roIe as specific sugar carrier on artificial lipid membranes [7].Rabbit small intestinal sucrase isomaltase complex, as obtained by papain solubilization and Sephadex G-200 chromatography, has a molecular weight of approximately 220000 [2,3,8] (and Mosimann, H. and Semenza, G., unpublished results). As a further step towards its chemical characterisation we report here a simplified isolation procedure and data on its chemical composition. EXPERIMENTAL PROCEDURE Amino-Acid CompositionPrior to hydrolysis the protein samples were dialyzed against water. Their concentrations were and as confirmed by us, referring to the weight of protein samples which had been exhaustively dialyzed and dried under vacuum a t 80 "C in a "drying pistol"). Duplicate analyses were made with hydrolysis times of 24, 48, 72, 96 h, respectively. The protein samples (500 pg) were hydrolyzed in 2 ml 6 N HCl under vacuum a t 110 "C, norleucine being used as internal standard. The amino acids were determined on a BC-200 Analyzer from Calbiochem.Tyrosine and tryptophan were determined by the methods of Bencze and Schmid [lo] Total half-cystine and cysteine was determined by four different methods. Firstly, with 5,5'-dithiobis-(2-nitrobezoic acid) according to Ellmann [la] after denaturation and reduction of the protein in 8 M urea with 2-mercaptoethanol (65 nmol/mg protein at 37 "C for 1. h under nitrogen). Secondly it was determined as aminoethyl-cysteine [15] ; in this method several experiments were made using different concentrations of guanidine hydrochhride or urea as denaturating agent and mercaptoethanol or dithiothreitol as reducing agent. The highest yield was ob...

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Section: 98supporting

confidence: 82%

A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition

Cogoli

Mosimann

Vock

et al. 1972

European Journal of Biochemistry

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“…The presence of various species of lysozyme activities within the connective tissue extracts was not unexpected since multiple forms of lysozyme have been reported to be present in other mammalian cartilages^. The finding of lysozyme activity in the low M r pool from costal cartilage with an apparently low pi questions the classification of these proteins as lysozymes using the criteria proposed by Jolles [21] .…”

Section: Discussionmentioning

confidence: 94%

A Comparative Study of the Low-Molecular Mass Serine. Proteinase Inhibitors of Human Connective Tissues

Andrews¹,

Melrose²,

Ghosh³

1992

Biological Chemistry Hoppe-Seyler

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Low molecular mass serine proteinase inhibitors isolated from human articular cartilage, intervertebral disc, meniscus, and costal cartilage were compared chromatographically. Similar charge and size properties were exhibited when these inhibitors were examined by gel permeation and cation exchange chromatography. The individual proteinase inhibitory species separated by these procedures all cross-reacted with a polyclonal antibody raised against the mucous proteinase inhibitors (MPIs) obtained from human bronchial secretions, however the distribution of these MPI-like species varied with the origin of the connective tissue. The major inhibitory species present in human articular cartilage and intervertebral disc were purified to homogeneity using gel filtration, cation exchange, trypsin affinity and high performance reverse phase chromatography. The amino-terminal sequences of the purified cartilage intervertebral disc inhibitors was found to be identical to the published sequence of MPIs isolated from parotid and seminal secretions. These findings indicate that the endogenous small molecular mass cationic serine proteinase inhibitory proteins present in human cartilaginous connective tissues are members of the MPI family of proteinase inhibitors.

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“…Evidence for the overlaps between tryptic peptides is indicated in Table 3. [13] obtained a great number of chromatog;raphically pure lysozymes from normal human tissues and secretions (milk, tears, saliva, placenta, spleen, serum, leucocytes) by ion-exchange chromatography on Amberlite CG-50 : all these enzymes had the same chromatographic behaviour ; the main active peak, preceded by a small active fraction (< 5%), was eluted at the same place when the same column was employed. Multiple forms were not observed as in tlie case of the lysozymes isolated from duck egg-white [6].…”

Section: Peptidementioning

confidence: 99%

Human Milk Lysozyme: Unpublished Data Concerning the Establishment of the Complete Primary Structure; comparison with lysozymes of various origins

Jollès

1971

Helvetica Chimica Acta

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Sznnamary. Details conccrning the establishment of the coniplcte primary structure of h u m a n milk Iysozynie (prcviously published in a preliminary note) are presented The chymotryptic peptides obtained from thc reduced alkylated enzyme wcrc purified and their aniino acid sequences clctermined chiefly by thc 'Edman-dansylation' procedure, and in two cases by partial acid 01peptic hydrolyses. The tryptic peptides are alined into a single chain containing 129 amino acid rcsidues, on the basis of ovcrlapping pcptidcs. T\vo labile glutarninc: residues easily converted into glutaniic acid residues werc characterized. I-Tuiiian milk lysozyme is compared with other hutnan lysozymcs (from normal and leuchaemic individuals) prepared by our group. The structure proposed is idcntical with the sequence of human Ieuchaemia lysozyme (from the urine of a patient with chronic monocytic leuchaemia) reported b y Can.fzeld. H u m a n inilk lysozyme is also near by related to several bird egg-white lysozynies (and bovine cc-lactalbumin) : identical positions of Cys and Trp residuos and of the residues essential for the catalytic activity or involved in some hydrogcn honds; several identical rcgions, espccially in the /3-sheet region.; between 71 and 77 identical amino acid residues. I t is suggcstecl that by an insertion and a deletion in the sequence of human milk lysozyme, sequcnces homologous t o thosc of bird lysozymes can be obtaincd.

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Lysozyme: Ein Kapitel Molekularbiologie

Cited by 19 publications

References 110 publications

A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition

A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition

A Comparative Study of the Low-Molecular Mass Serine. Proteinase Inhibitors of Human Connective Tissues

Human Milk Lysozyme: Unpublished Data Concerning the Establishment of the Complete Primary Structure; comparison with lysozymes of various origins

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