H. Bloemendal

On the subunits of α-crystallin

α-Crystallin was isolated from total water-soluble lens extract by preparative zone electrophoresis on starch or Pevikon C870 blocks and purified by density gradient centrifugation and Sephadex chromatography. These preparations were treated with urea or sodium dodecyl sulphate and submitted to electrophoresis on polyacrylamide gels containing either urea or sodium dodecyl sulphate. Whereas in 7 M urea a large number of zones was detected, only three bands were observed in 1% sodium dodecyl sulphate. On the other hand, the sedimentation coefficient had the same value in both media. We re-investigated the N-terminal amino acid content in the starch block preparations and compared the result with that obtained from the preparations isolated according to our new procedure. The concentration of dinitrophenyl-glutamic acid was lower in the purified samples. Urea-treated samples had the same concentration of N-terminal glutamic acid whereas the concentration of the “trace” end-groups did not change. Sedimentation-diffusion equilibrium in the analytical ultracentrifuge revealed a slight heterogeneity in the purified samples. Electron micrographs of electrophoretic and of further purified samples were very similar. At neutral or slightly alkaline pH they showed almost uniform spherical aggregates in which a substructure was observed. At acid pH, coiled filaments rather than small globules could be demonstrated.

Studies on cytoplasmic ribonucleic acid from rat liver. I. Fractionation and function of soluble ribonucleic acid.

Abstract 1. 1. The soluble polynucleotides isolated by phenol extraction from rat-liver cytoplasm were fractionated on the resin Ecteola. 2. 2. Four fractions were obtained, one of which (s3-RNA) could be transferred to microsomal RNA in the presence of soluble enzymes, ATP, GTP and a nucleoside triphosphate regenerating system. The efficiency of this transfer was about 17% based on the isotope content of the added polynucleotides. 3. 3. On incubation of pH 5 enzymes with DL -[14C]leucine or a mixture of radioactive amino acids in the presence of ATP, s3-RNA became mainly labelled. Minor amounts of radioactivity, not exceeding 10% of that recovered in s3-RNA, were also detected in other fractions. 4. 4. The fractions were further characterized by ultracentrifugal analysis, dialysis and treatment with EDTA. 5. 5. The possible significance of these results for the mechanism of protein biosynthesis in rat-liver microsomes is discussed.

Studies on cytoplasmic ribonucleic acid from rat liver II. Fractionation and function of microsomal ribonucleic acid

Abstract 1. 1. Rat-liver microsomal RNA isolated by phenol extraction from deoxycholate-insoluble particles was fractionated on the resin Ecteola, yielding at least three fractions. 2. 2. Following in vitro incubation of microsomes with 32 P labelled soluble polynucleotides, AT ATP, GTP, and a nucleoside triphosphate regenerating system, only one microsomal RNA fraction became radioactive. 3. 3. This selective metabolic transfer of soluble RNA (or a derivative thereof) resulted in the attachment to microsomal RNA by covalent bonds. 4. 4. Ribonuclease activity in the ribonucleoprotein particles became manifest after exposure of the particles to 8 M urea. The presence of this enzyme is discussed in connection with the transfer reaction.

Studies on cytoplasmic ribonucleic acid from rat liver: V. Cofactor requirements for the metabolic interaction between soluble and microsomal ribonucleic acid

Abstract 1. 1. The metabolic transfer of nucleotide label from [32P]s-RNA to microsomal RNA has been studied in a system containing rat-liver microsomes and varying concentrations of the four nucleoside triphosphates. 2. 2. ATP and GTP stimulate the reaction, whereas the other nucleoside triphosphates are much less effective. Optimal conditions, however, vary from batch to batch of s-RNA. By contrast, the transfer of [14C]leucine from [14C]leucyl-s-RNA to microsomal protein requires GTP only. 3. 3. Incubation of s-RNA with microsomes in a reaction mixture devoid of cofactors reduces its metabolic transferring abilities as compared with s-RNA incubated in the presence of these factors. Evidence is presented in this and the subsequent paper that the structural integrity of the s-RNA terminus is a requisite for the metabolic transfer of nucleotide as well as for amino acid transfer to the microsomal template. It is suggested that GTP is specifically required for the interaction between s-RNA (or part of the soluble polynucleotides) and the microsomal template whereas the stimulatory activity of ATP may be ascribed to its capacity to restore the functional end of s-RNA, although a more specific function of the latter nucleotide cannot be excluded. 4. 4. The experiments further indicate that s-RNA following incubation with microsomes, has fully retained its ability to interact with microsomes, as judged by its amino acid carrier function as well as by its capacity to exchange nucleotide material with the microsomal template, provided that ATP and GTP are present during the transfer reaction.

Studies on cytoplasmic ribonucleic acid from rat liver. III. Further characterizations of soluble and microsomal ribonucleic acid subfractions as isolated on modified cellulose ion exchangers.

Abstract 1. 1. Soluble and microsomal RNA isolated from rat liver were further characterized on a variety of Ecteola cellulose resins differing in densities of binding sites. 2. 2. In particular one microsomal RNA subfraction, showing similar chromatographic behaviour as soluble RNA, was studied in detail. 3. 3. Microsomal RNA from rat liver was unable to accept amino acids when incubated with pH-5 enzymes and ATP, in contrast to the results of Smith et al. 4. 4. A rapid and efficient procedure for the isolation of soluble RNA, free of mono- and oligonucleotides, was worked out using gel filtration on Sephadex. 5. 5. The mechanism of polynucleotide adsorption unto cellulose-ion exchange resins has been discussed and indications regarding the homogeneity of the exchangers have been obtained.

Studies on hormones from the anterior pituitary gland I. Identification and isolation of growth hormone and prolactin from the “granular” fraction of bovine pituitary

Abstract The granular fraction from bovine pituitary glands isolated by differential centrifugation was shown to contain the bulk of growth hormone and prolactin. These hormones were identified with the aid of immunological techniques and disc electrophoresis. The granular fraction, although heterogeneous by electron microscopy, proved to be a suitable starting material for a new isolation procedure of these two hormones. Extraction of the 12 000 × g sediment followed by deoxycholate treatment of the remaining particulate material yielded two fractions containing the bulk of growth hormone and prolactin, respectively. Precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose columns yielded 1 to 2 mg of growth hormone (1.7 I.U./mg) and 3 to 4 mg of prolactin (12.5 I.U./mg) per gram wet weight of pituitary material. After each purification step hormone activity as well as any possible trace contamination with serum proteins was determined by immunological methods. Protein patterns were obtained with the aid of zone electrophoresis on polyacrylamide gels.

Studies on cytoplasmic ribonucleic acid from rat liver: VI. Tentative mechanism of the metabolic interaction between soluble and microsomal ribonucleic acid

Abstract 1. 1. The metabolic transfer of nucleotide label from [ 32 P]s-RNA to microsomal RNA has been followed over various periods of time. Cofactors and s-RNA have been replenished during incubation with rat-liver microsomes. 2. 2. It has been demonstrated that both ATP and GTP stimulate the transfer reaction, but in an independent fashion. These results have been interpreted by assuming that only s-RNA molecules carrying the intact terminal structure are able to interact, provided GTP is present. It is furthermore suggested that ATP restores the structural integrity of the s-RNA terminus, although a more specific function for ATP so far cannot be excluded. 3. 3. A tentative representation of the mechanism of the interaction between s- and m-RNA is given, taking into account these and earlier data. A cyclic exchange of an oligo- or polynucleotide is proposed, comprising the terminal part of the s-RNA chain. Possible implications for the process of protein biosynthesis in rat-liver microsomes are discussed.