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A new preparation method of immobilized urease discs for biomedical applications. which was a thin circular film (200 μm,50mmφ), was developed. The method was achieved by electron beam irradiation of polyethyleneglycol diacrylate monomers in the addition of paper disc and bean powder as protective substance for irradiation by which a denaturation of the enzyme by irradiation was effectively prevented. The immobilized enzyme disc with a high enzyme activity (remaining activity yield), about 90%, was obtained. The enzyme activity was varied by the preparation conditions such as the thickness of paper disc, monomer concentration etc. The enzymes were trapped near the surface of the disc to be easily reacted with substrate. The trapped state of the enzymes appeared to be affected by a hydrophilicity of the polymers.

To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.

NG, N G - Dimethylarginine ( unsym - DMA ) has now been found to be present in myosin prepared from developing leg muscle. Neither monomethylarginine nor sym - DMA (NG N ′ G - dimethylarginine ) could be detected in our preparations. Cultured muscle cell myosin contains up to four residues of this amino acid per 5 × 105 grams of protein. Myosins from leg muscle of chick embryos and neonatal rats contained less than two residues of unsym - DMA , while none was detected in adult chicken or rat myosins. Cardiac myosins from developing as well as mature animals lacked or contained very little unsym - DMA . Actin was devoid of this amino acid.

The size of the active site of carboxypeptidase-A was investigated by studying the kinetics of hydrolysis of peptides of L-alanine, D-alanine and L-phenylalanine, as well as of a number of their N-benzyloxycarbonyl, N-acetyl, N-phenylproprionyl and N-methyloxycarbonyl derivatives. From a comparison of the various kinetic parameters ( K m, kcat) it was concluded that the active site of this enzyme extends over about 18 A. The binding area can be divided into 5 “subsites”, each accommodating one amino acid residue (or blocking group) of the substrate. By comparing K m values of pairs of substrates containing either a methyl or a benzyl side-chain in equivalent positions, it was shown that the binding area as a whole has a larger affinity towards the aromatic residues. Substitution of a D-residue for an L-residue reduced kcat values rather than K m. In addition a remarkable affinity for the urethane-grouping located specifically at subsite S3 was found. A methyloxycarbonyl or benzyloxy — carbonyl group occupying this subsite caused a 5-fold increase in K m as compared with an acetyl or phenylproprionyl group, respectively. Methyloxycarbonyl or benzyloxycarbonyl groups in subsites S2 or S4 showed no such effect.

Pseudouridine kinase activity has been demonstrated in extracts of some pyrimidine auxotrophs of Escherichia coli . This enzyme has a pH optimum of 7, and requires ATP, Mg++, and either K+ or NH4+. Pseudouridine kinase is distinct from uridine kinase and deoxythymidine kinase. The presence of pseudouridine kinase and pseudouridylate synthetase activities in strains capable of growth on pseudouridine (Ψrd) as the sole pyrimidine source supports the following pathway for Ψrd utilization

Bone and dentine collagens are virtually insoluble in the solvents employed to extract native tropocollagen from soft tissue collagens. The decrease in the ease of extraction of the tropocollagen is generally considered to be due to an increase in the presence of crosslinked components ( see Piez, 1968 ; Bailey, 1968a for reviews ). Bone and dentine collagen must therefore be extensively inter molecularly crosslinked, but the chemistry of the crosslink is unknown. Glimcher and Katz, 1965 , Glimcher et al., 1965 suggested that non-covalent bonds are involved in the stabilization of bone. However a recent report by Miller et al. (1967) clearly demonstrated the presence of stable covalent inter - chain crosslinks in the denatured guanidine-HCl extract from bone. Veis and Schlueter (1964) similarly concluded that dentine was extensively crosslinked by stable covalent bonds, and proposed phosphatemediated ester bonds in addition to a system of periodate sensitive bonds. Our recent studies on the crosslinks of soft tissue collagens have demonstrated the presence of both labile and stable inter molecular crosslinks ( Bailey, 1968b ; Bailey and Peach, 1968 ). The present communication describes the isolation and identification of two covalent inter-chain crosslinks stabilizing bone and dentine.

Summary The primary structure of α-bungarotoxin isolated from the venom of Bungarus multicinctus was determined. It composes of 74 amino acid residues including ten half-cystines. Comparing the sequence with those of cobras and sea snake species, striking similarities can be found, especially between α-bungarotoxin and Naja nivea α-toxin, indicating 50 % sequence homology.

A novel method to construct a stable and uniform phospholipid membrane of large area and good manipulability is reported. Using the Langmuir-Blodgett (LB) technique, a monolayer of phospholipid can be transferred to filter paper. The electrical conductance across the pores of the lipid membrane is about 1.8 X 10(-9) S/cm2, corresponding to the conductance of 10(-7)-10(-10) S/cm2 reported for bilayer lipid membranes (BLM) of phospholipids. A scanning electron micrograph demonstrated that the phospholipid membrane on the filter paper was uniform.

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.

A method for transferring small DNA fragments from composite polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM)-paper is described. DNA fragments are separated by electrophoresis in polyacrylamide-agarose gels crosslinked with N,N′-diallyltartardiamide instead of N,N′-methylenebis-acrylamide. The crosslinks are cleaved by treating the gel with periodic acid after electrophoresis and the DNA is denatured with alkali. After neutralization, the single stranded DNA fragments are transferred to DBM-paper and detected by hybridization with labeled DNA probes. The procedure has been used to transfer and visualize unlabeled SV40 DNA fragments in the size range 28 to 1790 base pairs.

A sequence of 107 amino acids has been determined in the single chain of the major parvalbumin (pI 4.36) from hake ( Merluccius merluccius ) muscle as a result of studies on peptides obtained after tryptic, chymotryptic, peptic and cyanogen bromide cleavages of the protein.

Radioactive methionine was injected into the thorax of honey bees. After several hours, mitochondrial and supernatant proteins were prepared from thorax and hydrolyzed with pronase. In these digests labelled N-formyl- and N-acetyl-methionine were detected. The latter was about evenly distributed between the two fractions, while the bulk of formyl-methionine was present in mitochondrial protein. Control experiments exclude a bacterial origin of these acyl-methionines.

Extensive digestion of the basic trypsin inhibitor by thermolysin at 60–80° has been demonstrated by loss of inhibitory activity, increase in ninhydrin positive material and separation of the breakdown products by electrophoresis. Formation of the inhibitor-trypsin complex does not protect the inhibitor from digestion by thermolysin; both components are digested. Between 40° and 80° the inhibitor undergoes a reversible transition involving a slight conformational change; this may account for its susceptibility to thermolysin.

The isolation and sequence of active site peptides from glycogen phosphorylase b is described. These cysteine peptides become exposed to preferential labeling upon removal of pyridoxal 5′-phosphate (PLP) from the enzyme and they have the sequence Ala-Cys, Asx-Ala-Cys-Asp and Asx-Glx-Lys-Cys-Gly-Gly. The first two of these peptides appear to be derived from the PLP binding site of phosphorylase.

In vitro methylation of E. coli tRNAfMet fragments or their reconstituted molecule by rat liver methylase showed that (1) the whole tRNA molecule was not necessary for recognition by tRNA methylase, and (2) the extent and the site of methylation of fragments differed depending upon the type of fragment used. This indicates that the conformational structure of the methyl acceptor molecule is important for recognition by the tRNA methylase.

An unknown polyamine isolated from a pseudomonad which contains no spermidine is identified as 2-hydroxyputrescine by comparison of the IR, NMR and mass spectra of its benzoylated derivative with that of the benzoyl derivative of 2-hydroxyputrescine synthesized from 1, 4-diamino butanone.