Enzymic proteolysis

Abstract

TRYPSIN-RESISTANT phosphopeptones from casein have been studied by Posternak [1927; 1928] and by Rimington [1927, 1, 2]. The former isolated three compounds which he designated 'lactotyrins' and showed them to be made up of 15 to 18 amino-acid units containing serine, glutamic acid, aspartic acid and isoleucine. Posternak's papers contain no experimental details, and although his conclusion that the P in casein exists in the form of phosphoserine has been confirmed by the isolation of this amino-acid by Lipmann [1933], the generally accepted view of the structure of phosphopeptone itself is that advanced by Rimington [cf. Jordan Lloyd & Shore, 1938], according to whom the enzymeresistant polypeptide is made up of 9 hydroxyamino-acid units consisting of 3 mol. of hydroxyglutamic acid, 4 mol. of hydroxyaminobutyric acid and 2 mol. of serine. The discrepancies in the results obtained by these authors and the fact that Harington & Randall [1931] were unable to identify hydroxyglutamic acid as a constituent of casein prompted the reinvestigation of this problem. To obtain a trypsin-resistant phosphopeptone of constant composition advantage was taken of the fact that peptic digestion of casein automatically causes the separation of an insoluble complex, the so-called 'paranuclein', which contains 50-60 % of the P of casein and only about 20 % of its N. After digesting the parannclein till constancy of amino-N was reached, the P-containing complex was precipitated with neutral lead acetate, redigested with trypsin and the phosphopeptone finally separated as the Ba salt insoluble in 50 % alcohol. Preparations so obtained were constant in composition and unaffected by further treatment with trypsin. The amino-N value of 10 % and the atomic N/P ratio of 3-2 to 3-3 indicated a polypeptide of 10 amino-acids attached to 3 phosphoric acid residues. N distribution determined according to Damodaran [1931, 1] gave amide-N values varying from 10 to 13 % in different analyses, dicarboxylic acid-N 43-47 % and monoamino-N 43-45 %, and a complete absence of basic

A phosphoprotein phosphatase from ox brain

“…Casein phosphopeptones. These were prepared by the digestion of a-casein with crystalline pepsin by the method of Damodaran & Ramachandran (1941). The yield from 1 g. of a-casein was 25 mg. of peptone containing 1% of phosphorus.…”

The estimation of threonine and serine in proteins

“…These corrections have accordingly been applied to the protein analyses given later in Tables 3 and 4, yet it should be emphasized that in these cases the application may not be strictly valid, since during the early stages of hydrolysis the hydroxyamino-acid concemed will be still in peptide combination, and hence perhaps more prone to decomposition than when in the free state. It is known that when serine is present in the protein in the form of an ester with phosphoric acid-as it is, for instance, to a small extent in casein-it undergoes considerable destruction on acid hydrolysis (Posternak, 1927;Damodaran & Ramachandran, 1941).…”

Preparation and amino acid composition of enzymically dephosphorylated casein

“…These values include corrections made for the decomposition of serine during acid hydrolysis of the protein. The corrected values for casein may be low, in view of the greater lability to acid hydrolysis of serine combined as phosphoserine in phosphoproteins, as compared with that of free serine (Damodaran & Ramachandran, 1941;Nicolet, Shinn & Saidel, 1942;Mecham & Olcott, 1949). Thus Nicolet et al (1942) give 7-38 % as the probable serine content of casein, compared with 5-5% found on direct acid hydrolysis.…”

“…The results obtained in the present investigation are in conformity with this suggestion. Additional evidence for this view is provided by the fact that the peptides formed during the dephosphorylation of casein contain, as common constituents, glutamic acid, isoleucine and serineamino acids which are also present in the phosphopeptones obtained from casein by various workers (Rimington, 1941;Damodaran & Ramachandran, 1941).…”