Hava Neumann

Computerized localization of peptides in proteins of known sequence

Abstract 1. 1. A numerical method for the location of peptides within the known sequence of a protein is described. 2. 2. The method consists of three steps: (1) enzymic or chemical cleavage of peptide bonds in the protein; (2) separation of the peptides by two-dimensional high-voltage electrophoresis followed by amino-acid analysis of the eluted peptides; (3) location of given peptides in the sequence by computer, using only some of the amino-acid composition of the peptide. 3. 3. The advantage of the computer program (last step), apart from the great speed at which results are obtained, is the allowance which is made for imprecision in the information regarding certain amino-acids, e.g., glycine, serine, tryptophan, methionine, tyrosine, etc. 4. 4. Furthermore, from the information obtained from the two-dimensional high-voltage electrophoresis, the computer program determines whether a carboxylic acid side chain is present in the acid form or as its amide. 5. 5. The method has been tested successfully using tryptic digestion of derivatives of lysozyme and ribonuclease.

Photoreactions of phosphorothioate and cysteamine-S-phosphate: Photosubstitution and photophosphoryl transfer

The photoreactions of phosphorothioate and cysteamine-S-phosphate were investigated. On irradiation of phosphorothioate a marked change in absorption spectrum was observed. The product migrated in high voltage electrophoresis, with different mobility from that of phosphorothioate and its dimer, or inorganic orthophosphate. It contained phosphate and sulfur in a ratio of 2: 1, without reducing properties. Therefore it was suggested that the product is either pyrothiophosphate, or a cyclic compound, with similar composition. On irradiation of phosphorothioate in the presence of potential phosphoryl group acceptor, such as glucose or galactose, 25-40% of the phosphoryl group was transferred. The formation of glucose 6-phosphate, or galactose 6-phosphate was observed. The photolysis of cysteamine-S-phosphate gave cysteamine, inorganic orthophospate and taurine. Under the same conditions of irradiation, inorganic orthophosphate or aminoethanol-O-phosphate were found to be stable.

Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolyme of ethylene and maleic anhydride

Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50–70% of its optimum activity in the pH range 7–8, while APase retained only 5–15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S20,w values. S20,w values obtained for APase-EMA, activated APase, and APase dialyzed against wafer were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, Mr, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. Mr values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S20,w to 5.15 and Mr to 82,000.

Some physical properties of alkaline phosphatases found in various tissues of AKR and C57BL/6 mice, normal and leukemic

The appearance and levels of alkaline phosphatases in the thymus of AKR and C57BL/6 leukemic and normal mice have been investigated mainly histochemically [l-3] . As far as we are aware no systematic study of the quantity and kinetic behavior of the alkaline phosphatases of the thymus, the mesenteric nodes, the spleen, the liver, and the kidney derived from normal and leukemic tissues of mice has been reported. In this publication we describe the enzymatic hydrolysis of the substratesp-nitrophenyl phosphate @NPP) and cysteamine S-phosphate (CASP) by alkaline phosphatases extracted from various tissues of normal and leukemic mice. Quan itative determinations of the rate of hydrolyses of the above substrates were studied under different experimental conditions such as pH and the concentration dependence on MgC12, NaCl, in tris, and in sodium barbital buffers. The data obtained were compared with the corresponding data using alkaline phosphatases from the chicken intestine and Escherichia coli under similar conditions either described in the literature [4-61 or obtained in this study.

Numerical technique using a digital computer for the determination of the specificity of proteolytic enzymes and chemical cleavages

Abstract 1. 1. A numerical technique is presented for the determination of the specificity of proteolytic enzymes and chemical cleavages. The technique was tested using trypsin as the proteolytic enzyme and reduced and alkylated derivatives of lysozyme and ribonuclease as substrates. 2. 2. The number and charge of the tryptic peptidcs (separated by high-voltage electrophoresis) as well as the number of tryptophanyl or tyrosyl peptides, the linear sequence, and the charge at pH 6.5 of each amino-acid residue of the model protein tested were supplied to the computer. 3. 3. The data obtained from the computer with the aid of the above information were in agreement with the known specificity of trypsin, and the composition of each peptide derived by the computer was identical with those obtained by direct analysis of the peptides eluted from the clectrophoretograms.