Pierre Jollès

The amino acid sequence of sheep κA-casein: II. Sequence studies concerning the κA-caseinoglycopeptide and establishment of the complete primary structure of the protein☆

The primary structure of sheep κA-caseinoglycopeptide, the C-terminal portion of fκA-casein released by chymosin, was established. The peptide was subjected to digestion with chymotrypsin and the chymotryptic peptides were purified by Sephadex G-50 filtration and Dowex 1X2 chromatography; their sequences were determined with a Sequencer. Alignment of the chymotryptic peptides into a chain containing 66 amino acids was possible by automated degradation of the caseinoglycopeptide up to the 45th amino acid. This study allowed the establishment of the complete sequence of sheep κA-casein (171 amino acids) which is indicated. For the first time a comparison between two κ-caseins (cow, sheep), substrates of chymosin (milk clotting process), was possible. Two deletions and 26 replacements, especially in the caseinoglycopeptide part, were noted, and the location of the carbohydrate part was indicated. Some structural features concerning the caseinoglycopeptides and the fibrinopeptides involved in the milk and blood clotting processes are briefly compared. Three examples of sequences of portions of cow or sheep κ-caseins and of human fibrinogen presenting homology are presented.

Amino acid sequence of guinea-hen egg-white lysozyme☆

Abstract Guinea-hen egg-white lysozyme was obtained in a chromatographically pure state by ion-exchange chromatography on Amberlite CG-50. The reduced alkylated enzyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1-X2 chromatography and electrophoresis and chromatography on paper. The amino acid sequence of these peptides was determined chiefly by the Edman dansylation procedure. Alignment of the tryptic peptides into a single chain containing 129 amino acids was determined from basic overlap peptides obtained by digestion of the enzyme with chymotrypsin. 10 replacements were noted between guinea hen and hen lysozymes. Further comparisons were achieved with 4 other bird egg-white lysozymes the structures of which were already known.

Comparison between human and bird lysozymes: Note concerning the previously observed deletion

We recently published the primary structure of human milk lysozyme (EC 3.2.1.17) [ 1,2] . In order to optimize homologous relationships, an insertion and a deletion were suggested to occur in the sequence of human lysozyme when compared to bird lysozymes [2,3]. The most probable position of the insertion seemed to be residue no. 48 and the corresponding tryptic peptide was studied in detail (tryptic peptide no. 5b; for the numbers of the peptides quoted in this paper, see [l] or [2]). From considerations of homology [ 1,3,4] the deletion was localized after residue no. 100. The primary structure of human leukaemia lysozyme isolated from the urine of a patient with chronic monocytic leukaemia [3] was similar to that of human milk lysozyme [ 11. Quite recently, Thomsen et al. [5] were able to demonstrate by an elegant method (personal communication; to be published) that human leukaemia lysozyme does not show a deletion after residue no. 100 when corn; pared to bird lysozymes. The peptide beginning from residue no. 98 contained the sequence: Arg-Val-Val-Arg-Asp-Proindicating that there are 2 residues of Val (and not only 1) between the 2 residues of Arg. It seemed important to reinvestigate the corresponding sequence of the normal human milk lysozyme, more especially because lysozyme from tissues or secretions of leukaemia patients (but not of healthy persons) contains two main active lysozyme fractions which were characterized by ion-exchange chromate graphy on Amberlite CG-50 [6]. Furthermore preliminary results on the structure of baboon milk lysozyme

A hydrosoluble, adjuvant-active mycobacterial “polysaccharide—peptidoglycan”. Preparation by a simple extraction technique of the bacterial cells (strain Peurois)

Mycobacteria possess several important biological properties as an adjuvant effect in antibody formation (Freund’s adjuvant effect) [I] and the induction of experimental arthritis in rats termed adjuvant arthritis [2]. Cell walls from human and non human mycobacterial strains as well as waxes D with a nitrogen-containing moiety (peptido-glycolipids) provoke these reactions [3,4] . In the different active substances, a polysaccharide (Poly, mainly an arabinogalactan [5] ) is linked to a peptidoglycan (PA) 161. Poly-PA constitutes the hydrosoluble moiety which can be joined by ester linkages to the lipidic part (Lip, mycolic acids as in wax D). We studied previously in detail the nitrogen containing part of waxes D from human origin [6,7] and we could thus demonstrate a close relationship between the peptidoglycan of an active wax D and the material which constitutes the backbone of mycobacterial as well as other cell walls. Several procedures (saponification IS], acetylation [9] ) allowed to obtain the hydrosoluble moiety, but nearly all of them modified more or less the sugars, especially the amino sugars, as established by analytical methods. Only the homogenization of wax D in a buffer medium [lo] seemed to yield a “native” Poly-PA, i.e. a hydrosoluble moiety where the sugars remain unchanged during the preparation steps. Our chemical studies strengthened the view that wax D could be some degradation product of the

The lysozyme from Nephthys hombergi (annelid)

Lysozyme (Mucopeptide N-acetylmuramylhydrolase, EC 3.2.1.17) from Nephthys hombergi (Annelid) was obtained in a chromatographically and electrophoretically pure state by gel filtration and ion-exchange chromatography on Amberlite CG-50. The quantitative amino acid composition, the molecular weight and the N-terminal amino acid of this enzyme are reported. Its heat stability and its activity as a function of pH at constant ionic strength were studied and compared to those of hen egg-white lysozyme.

Comparative study of cow and sheep κ-caseinoglycopeptides: Determination of the N-terminal sequences with a sequencer and location of the sugars

the casein rniccile in its natural etivironment 111 and in-the clotting phenomenon induced by t&action of rennin (EC 3.4.4.3); it’ is also the only casein fraction hi cOIlti3iM sugars 12, 31, TO the hCtCXOgC&ty Of K-cascin from pooled milk are contributing the genetic variants, but also the non-identical composiPion of the carbohydrate groups present. This observation is in ~cordance with Cottschalk’s [4] concept on the hetcrogtineity of the eaibohydrate group iti glyeoprot&s_ During the rennin clotring of milk a. Phe-Met bond ,is split J5]. A large peptide, called K-macropepride wheri K-case+ is sugar-free or K-caseinoglycopeptide when K-casein is sugar-rich, is liberated and paraK-casein, .devoid of sugars, precipitates. Bovine K-caseino&copepticle has already been submitted to extensive structural studies [6-91 but only few data are available foi sheep K-caseinoglyc&peptide [5, IO, I1 ]_ The prcsqtt note iepcrts the deter&nation of the E-terminal sequence of the latter (48 amino acid residues) with a sequencer and some comparisons with cow Ec-chseinogllycopept?de; furthermore the locatic~~ of the short giycopeptide obtained indepe&entty Corn cow #-casehi by enzymic procedures is iliscvssed. Cow KA-casein was prepared according to McKenzie and Wake [ 12J from the milk of homozygous cows and sheep K-casein according to AIais and Jollih [ 1.3]_ The K-c%einoglycopeptides were obtained after rennin digestion of the corresponding K-caseins as previously described [ 141, Automated Edman degradation [ 151 was carried out in a E&man sequencer, Model

THE TURNIP LYSOZYME

90 E, by the quadrol method. The thiazolinones wer;: converted into PTH-amino acids and these latter were characterized by thin-layer chromatography, by gas-liquid chromatogiaphy (Beckman GC 45 chromatograph) or with an amino acid autoanalyzer after regeneration of the free amino acid_ Short glycopeptides were isolated from cow and sheep K=% casein= by enzymic digestions <neBeaminidase, EC 3_2_ I. 18; chymotrypsin, EC 3.4.4.5; pronase) and further purified by filtration on Sephadt-x C-25 and paper electrophoresis according to Fiat et al. [ 16]_ Their structures were established mainly by the manual Edman technique.

Amino acid sequence of lysozyme from baboon milk

Soon after his discovery of ‘lysozyme’ (EC 3.2.1.17). Fleming detected the presence of this enzyme in many vegetable tissues, especially in the flowers [l] . A number of roots and tubers were also examined [2] : of these the only one which showed any marked bacteriolytic effect was the turnip. In comparison to human tissues, the lysozyme content of turnip was weak, but a 1 in 10 extract completely cleared an opaque suspension of Micrococcus lysodeikticus in one hr [2] . Since Fleming’s observations, only a very small number of studies have been devoted to plant lysozymes, and these all deal with the enzyme contained in latex. Meyer et al. [3] first reported lysozyme activity in crude proteolytic enzyme preparations from papaya and fig latex. In 1955, Smith et al. [4] prepared a crystalline lysozyme from papaya latex and quite recently Howard and Glazer [5, 61 studied such a lysozyme in detail. It was found that papaya lysozyme had a molecular weight of about 25,000, that glycine was its sole N-terminal as well as C-terminal residue, and that the molecule consisted of a single polypeptide chain. Papaya latex lysozyme lysed M. Zysodeikticus cell walls at one third of the rate exhibited by hen egg-white lysozyme; whereas papaya lysozyme ex. hibited 200-400 times higher chitinase (EC 3.2.1.14) activity toward chitotetraose than hen egg-white lysozyme, with whole chitin as substrate, it was only 10

κ-Casein from bovine colostrum

Abstract Reduced alkylated baboon milk lysozyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1 (X2) and chromatography and electrophoresis on paper. The amino acid sequence of these peptides was determined in detail chiefly by the Edman procedure. Alignment of the tryptic peptides into a single chain containing 130 amino acids was established chiefly by homology with human milk lysozyme; 14 replacements were noted between the two enzymes. Baboon milk lysozyme was devoid of methionine and contained six basic amino acids (arginine residues) less than human milk lysozyme.

An extracellular rennin-like enzyme produced by Physarum polycephalum

Abstract Whole casein from colostrum contains more κ-casein than normal casein. Structural studies have shown that the caseinoglycopeptide (C-terminal moiety) of κ-casein seems to be already present at parturition. Colostrum κ-casein contains about two times more carbohydrate than normal casein and the presence of N-acetylglucosamine together with N-acetylgalactosamine suggests structural differences of its prosthetic group. The behaviour of the sugars after parturition is discussed.