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1. A low-molecular-weight and a high-molecular-weight 1,4beta-glucan glucanohydrolase (Cx enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-Sepharose 6 B) and isoelectric focusing. 3. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. 4. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogenous in sedimentation equilibrium analysis. 5. The molecular weights of the enzymes were 12 500 and 50 000 +/- 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. 6. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10 degrees C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. 7. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard.

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.

1 A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ɛ-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2 The technique has been successfully tested with insulin. 3 When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4 A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

Starting from 2-thiodeoxythymidine, the mono- and triphosphate of the nucleoside analog were prepared by enzymatic phosphorylation with thymidine kinase and thymidylate kinase, respectively. 2-Thiodeoxythymidine triphosphate was also obtained by chemical pyrophosphorylation of the monophosphate. The analog nucleoside-triphosphate is a substrate for Escherichia coli DNA polymerase. A copolymer consisting of alternating deoxyadenosine- and 2-thiodeoxythymidine nucleotides is synthesized extensively by the enzyme. The polymer was characterized by nearest neighbour analysis, sedimentation and buoyant density in the analytical ultracentrifuge, ultraviolet spectra and stability against thermal and alkaline denaturation.

From the lipopolysaccharides of two Escherichia coli strains (serotypes O 7:K1:(L):H− and O10:K5 (L):H4) two unusual amino sugars were isolated. By sequential oxidation with periodate and hypoiodite, followed by identification of the reaction products, the aminosugars were characterized as 4-amino-4,6-dideoxy-d-hexoses. The chromatographic properties of the amino sugars and their N-acetyl derivatives were compared with those of authentic samples of free and N-acetylated 4-amino-4,6-dideoxy-d-glucose and 4-amino-4,6-dideoxy-d-galactose. On the basis of the results obtained the unknown amino sugars were tentatively identified as 4-amino-4,6-dideoxy-d-glucose (from the lipopoly-saccharide of E. coli O7:K1:(L):H−) and 4-amino-4,6-dideoxy-d-galactose (from the lipopolysaccharide of E. coli O10:K5(L):H4). This is the first time that 4-amino-4,6-dideoxy-d-galactose is described as a constituent of a polysaccharide.

An acidic polysaccharide was isolated from wild type (K+) Escherichia coli 09:K29 (A) but could not be isolated from an uncapsulated (K−) mutant of this strain. Analytical ultracentrifugation showed that the polysaccharide was physically heterogeneous but became homogeneous when treated with alkali. Alkali treatment also reduced the viscosity of aqueous solutions of the polysaccharide, while other physical properties were not affected. The molecular weight of the alkali-treated acidic polysaccharide is 332000, as calculated from s20 (4.7 × 1013 sec) and D20 (1.08 nm/sec). The polysaccharide consists of glucose, mannose, galactose and glucuronic acid in the molar ratios of 2:2:1:1. It also contains an acid labile substituent which was tentatively identified as pyruvate. After partial hydrolysis 7 oligosaccharides were isolated and analyzed. Oligosaccharide analysis, together with the results of methylation, periodate oxidation and carboxyl reduction indicated that the acidic polysaccharide consists of the hexasaccharide repeating unit The glycsoidic bond between the repeating units is an α-galactosyl-(1,6)-mannose linkage. The alkali-treated acidic polysaccharide represents a subunit chain with a length of about 300 repeating units. It is suggested that these subunit chains are interlinked by alkali labile ester bonds between the carboxyl group of glucuronic acid residues and hydroxyl groups in the chains. With the aid of passive haemagglutination and immune precipitation in O-, OK-, and K sera and with bacterial agglutination in OK serum before and after absorption with the acidic polysaccharide it was shown that the acidic polysaccharide represents the K29 antigen. Serological studies with chemically altered polysaccharide preparations indicated that glucuronic acid and mannose are immunodominant sugar residues. Inhibition of the K29 specific precipitin reaction with oligosaccharides obtained from the K29 polysaccharide and with the degraded polysaccharides of Salmonella anatum and Salmonella newington indicated that the determinant region of the K29 antigen is represented by the trisaccharide Man, which is probably partially substituted by pyruvate.

Mild acid hydrolysis of the glycolipid derived from a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and l-glycero-α-d-mannoheptosyl-1,5-KDO. By the use of various methods of KDO determinations, and by periodate oxidation performed on the glycolipid, results were obtained which indicate that the bridge between the polysaccharide portion of lipopolysaccharides and lipid A is formed by a branched KDO trisaccharide whose KDO side chain is randomly substituted by phosphorylethanolamine according to the formula —Hep-1,5-KDO-KDO-lipid A | KDO-(phosphorylethanolamine) Analogous results were obtained with P-lipopolysaccharides of other Salmonella mutants.

The reaction of phosphorothioate with bovine pancreatic ribonuclease at pH 9.0 in the absence of urea led to a derivative, denoted 4PS-ribonuclease, in which two of the four disulphide bonds in the molecule were opened. 4PS-ribonuclease is a unique molecular species, and not a mixture of native and open-chain ribonuclease; this is apparent from electrophoretic studies. By the use of the “diagonal map” technique it has been established unequivocally that the disulphide bonds opened were between half-cystines 3–8 and 4–5, counting from the amino terminus along the ribonuclease chain. 4PS-ribonuclease is fully active enzymically toward RNA, and more active than the native enzyme toward cytidine 2′,3′-cyclic phosphate. It is indistinguishable from native ribonuclease in its immunological reaction with antiserum to ribonuclease, and in its spectrophotometric titration, which shows that three of the six tyrosine phenolic groups in 4PS-ribonuclease titrate abnormally. Unlike native ribonuclease, 4PS-ribonuclease is digested by trypsin, even though at a lower rate than open-chain ribonuclease.

The λ-chains of normal pig γG-immunoglobulin were digested with pepsin in 1 M formic acid. Cystine-containing peptides were isolated by gel filtration on Sephadex G-25 and by chromatography on Dowex 50 and Dowex 1. The following structures were found: In addition to these peptides obtained in a high yield similar peptides were found but their complete structures were not determined in view of their low yield. The structures found in the λ-chains display a high degree of similarity with corresponding structures known to exist in human Bence-Jones proteins of the l-type.

Nitration of pancreatic trypsin inhibitor with tetranitromethane affords a mono-and a disubstituted derivative which was shown by sequential analysis to contain nitrotyrosine in position 10 and in positions 10 and 21, respectively. Both retain full trypsin inhibitor activity. Measurements of optical rotatory dispersion in the 230 nm region indicate that the secondary structure of the protein is unaffected by nitration. The nitrated derivatives exhibit a conformation-dependent side-chain Cotton effect in the region of nitroaromatic absorption. No such Cotton effect is shown by a nitrotyrosine-containing pentadecapeptide isolated from the tryptic digest from the nitrated and oxidized inhibitor.