Search

The electrophoretic behaviour of oligodeoxynucleotides on ion-exchange papers was studied systematically with particular attention to applications in sequence analysis. Complex mixtures of larger oligonucleotides were fractionated by electrophoresis on cellulose acetate followed by t.l.c. on polyethyleneimine-cellulose. The behaviour of the oligonucleotides on polyethyleneimine-cellulose and on the two-dimensional system can provide a useful guide to their sequence. Detailed results from some of these experiments have been deposited as Supplementary Publication SUP 50031 (13 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

Dimensionless apparent ionization constants of charged low-molecular-weight species may be obtained from paper-electrophoretic data at 20-25 degrees C with buffers (I0.1-0.5) of measured pH (1.5-12.5) containing oxalate ions. Relative mobilities rather than absolute mobilities were measured by using glycerol and m-nitrobenzenesulphonate respectively as standards of zero and unit mobility. Application of the procedure to ionizations of adenine, adenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, 3′:5′-cyclic AMP, ADP, ADP-glucose-agrocin 84 and ATP is described.

RESP-3032

1. A new method is described for f∈≥rpr∫∈g'cysteicaceptsderivedomthedisϕ––debrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisf∈≥rpr∫∈g′cysteicaceptsderivedomthedisϕdebrsofprote∈s.Cyst∈epeptsareseparatedbypapere≤ctrophoresisand⊗zedonpaperbyperformicacapour.E≤ctrophoresisatright∠s→thefirstdirection∏ucesparal≤lgroupsofcysteicaceptsly∈goffadiagonal.Thisfingerprint' reveals the way in which the cysteic acid peptides were originally joined in the protein. 2. The method allows a very easy selective purification of cysteic acid peptides. 3. By applying this method to bovine chymotrypsinogen A, we found that the half-cystine residues were linked 1–122, 42–58, 136–201, 168–182 and 191–220.

A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.

The Medical Research Council Medicinal Plants Survey in its expedition to Africa collected many specimens of the seed of Strophanthus hi8pidus P.DC. and S. sarmento8us P.DC., mostly from individual plants, in order to investigate the occurrence of sarmentogenin, a substance which forms a possible starting material for the production of cortisone. The isolation of sarmentogenin from certain of these seeds has already been reported (Callow, Meikle & Taylor, 1951). The present paper is a report on the preliminary investigation ofall the samples collected, and reveals certain interesting features about the distribution of cardiac aglycones in various Strophanthus species. As the examination of each seed sample by isolation of the constituents might take several years for completion, a method has been developed for the routine semi-micro investigation of the easily hydrolysable glycosides of the seeds. These easily hydrolysable glycosides (Reichstein, 1951), which are completely hydrolysed by refluxing for half an hour in 50% aqueous methanol which has been made 0-1N with respect to sulphuric acid, are those in which the steroidal portion of the molecule is directly attached in a glycosidic linkage to a 2deoxy sugar. Most of the best known glycosides belong to this group, which is much easier to investigate than the group in which the aglycones are directly attached to 'normal' sugars carrying a hydroxyl group at C(2), such as glucose or rhamnose, where isolation of the intact aglycone is usually difficult and has in several cases not yet been achieved. Investigation of the 'normal' glycosides in our seed samples has so far only been carried out in selected cases. The paper-chromatographic method of Zaffaroni, Burton & Keutmann (1950) (cf. Burton, Zaffaroni & Keutmann,1951)hasbeenusedbySchindler & Reichstein (1951) for separating this type of substance, but the procedure is in many ways complicated

1. A diagonal electrophoretic technique for studying the amino acid sequence around cysteine and cystine residues in proteins is described. The residues are first converted into S-aminoethylcysteine, and the protein is then treated with S-ethyl trifluorothioacetate, which trifluoroacetylates all the protein amino groups. The modified protein is digested enzymically and the resulting peptides are separated by paper electrophoresis. After exposure of the peptides on the paper to ammonia vapour, the electrophoresis is repeated, this time at right angles to the original direction. Peptides from which a trifluoroacetyl group is removed by the ammonia treatment will vacate the 45° diagonal formed by all other unaffected peptides owing to the exposure of an additional amino group and consequent increased electrophoretic mobility towards the cathode. Peptides containing lysine or S-aminoethylcysteine are readily purified by this technique. 2. The successful application of the technique to bovine insulin is described. 3. Various methods for distinguishing peptides containing lysine from those containing S-aminoethylcysteine in more complicated proteins are suggested and discussed.

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s−1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.

Although myo-inositol is included in media for the successful growth of plant tissues, the actual requirement of most tissues, including soybean (Glycine max) callus in suspension culture, for myo-inositol has not been demonstrated. We have made use of deoxyglucose to reduce intracellular levels of myo-inositol. Deoxyglucose is phosphorylated to deoxyglucose 6-phosphate, which inhibits L-myo-inositol 1-phosphate synthase, an important enzyme in the synthesis of myo-inositol. Addition of deoxyglucose to the medium resulted in a decrease in the intracellular level of myo-inositol that corresponded with a decrease in cell division. Cell viability was not affected. When myo-inositol was added to cells along with deoxyglucose, cell division was restored, as were intracellular levels of myo-inositol. Addition of myo-inositol had no affect on the uptake or metabolism of deoxyglucose. From these results we propose that myo-inositol has a role in maintaining cell division in soybean callus tissue in suspension culture.

1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

1. The renin-like substance isolated from the placenta of the rabbit produces a prolonged increase of blood pressure in the nephrectomized rat. If incubated with renin substrate from ox serum, it forms a pressor substance that elevates blood pressure in exactly the same way as does angiotensin. 2. This angiotensin-like principle was concentrated by means of ion-exchange chromatography and compared with Val-5-angiotensins I and II in two paper-chromatography systems and in paper electrophoresis. 3. In all the three methods the unknown principle behaved like a mixture of the two reference compounds. 4. It is concluded that incubation of the renin-like substance of placental origin with substrate from ox serum gave a mixture of Val-5-angiotensins I and II. This is evidence that the enzyme isolated from the placenta is either closely related to, or identical with, renin.