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1. Three peptides containing alpha-aminoadipic acid and cysteine have been obtained in small amounts from the mycelium of a Cephalosporium sp. 2. The peptides were precipitated as cuprous mercaptides together with glutathione and resolved from the latter and from each other by preparative paper electrophoresis and chromatography either in the sulphonic acid form or as S-sulphonyl derivatives. From the S-sulphonyl derivatives they were obtained in the thiol form. 3. One peptide (P3) was shown by amino acid analysis and the mass spectrum of the NS-ethoxycarbonyl derivative of its methyl ester to be delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine. A second peptide (P2) contained alpha-aminoadipic acid, cysteine, valine and glycine, and the third peptide (P1) contained alpha-aminoadipic acid, cysteine, beta-hydroxyvaline and glycine.

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[(14)C]valine was added to a shaken suspension of the organism. More (14)C was incorporated into peptide P3, delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing alpha-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing beta-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of delta-(l-alpha-aminoadipyl)-l-cysteine and dl-[(14)C]valine. No synthesis was observed in the presence of delta-(d-alpha-aminoadipyl)-l-cysteine or of dl-alpha-amino[(14)C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.

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SUMMARYOsmotically fragile protoplasts have been prepared by the action of lytic enzymes on the mycelium of Penicillium chrysogenum and Cephalosporium acremonium.The yield of protoplasts, based on DNA content, was up to 18 %. Pretreatment of the mycelium with a thiol compound was necessary with Cephalosporium but not with Penicillium. Electron micrographs indicated that the protoplasts contained all the intracellular organelles of the mycelium and were bounded only by a cytoplasmic membrane. The metabolic activity of the protoplasts, as measured by their respiration, ability to maintain intracellular amino-acid pools, and antibiotic production, was similar to that of control mycelium. L-Valine and L-a-aminoadipic acid, which are precursors of penicillin N and cephalosporin C, were taken up and concentrated by the protoplasts of C. acremonium, although the latter transported L-valine less rapidly than did the corresponding mycelium. Protoplasts of P. chrysogenum took up ~-valine but not L-a-aminoadipic acid or its 8-ester. There was little or no transport of the corresponding D-amino acids by protoplasts of either organism. I N T R O D U C T I O NOne of the difficulties encountered in studies of penicillin and cephalosporin biosynthesis has been associated with the permeability barriers of mycelium. Thus, the interpretation of some experiments has been limited by the failure of a substance added to mycelial suspensions to reach the site of synthesis. For example, no detectable amount of [14C]penicillin N was found to enter the mycelium of Cephalosporium acremonium from the extracellular fluid (Smith, Warren, Newton & Abraham, I 967). The dipeptide 6-(a-aminoadipyl)-cysteine was not taken up intact by the mycelium but hydrolysed to its constituent amino acids (Loder, Abraham & Newton, 1969).A fragmented hyphal preparation has been obtained by ultrasonic treatment of the mycelium of Cephalosporium acremonium which is able to synthesize a possible biosynthetic intermediate, the tripeptide 8-(a-aminoadipyl)cysteinylvaline, from 6-(L-a-aminoadipyl)-Lcysteine and ~~-[ l~C ] v a l i n e (Loder & Abraham, 1971). However, no indication of penicillin or cephalosporin formation was obtained in fragmented hyphal systems of this type and it seemed that a study should be made of gentler methods of disrupting the mycelium. The first step of a possible approach was the enzymatic removal of the hyphal walls to form osmotically fragile protoplasts. For example, a bacterium-free extract was prepared from protoplasts of Bacillus licheniformis which was able to synthesize bacitracin from its constituent amino acids in the presence of an energy-generating system (Ishihara, Sasaki & Shimura, 1968), whereas an extract prepared by ultrasonication was less effective (Shimura, P. A. F A W C E T T A N D O T H E R SSasaki & Sugawara, 1964). This paper describes the preparation of protoplasts from strains of Penicillium chrysogenum and Cephalosporium acremonium and some of the properties of these protoplasts which are relevant to the biosynt...

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Summary. Glycolytic activity and nucleotide metabolism were studied in normal resting platelets and platelets exposed to collagen. Lactate production was found to be a more sensitive index of glycolytic activity than glucose consumption. In the presence of added glucose, the rate of lactate production was linear for at least 60 minutes. In the absence of glucose, lactate production continued for 60 minutes and appeared to be independent of glucose for the first 10–15 minutes. Addition of both collagen and pure tropocollagen to the platelet suspension produced a significant and constant increase in glycolytic rate. This was associated with release of ATP and ADP and a small but significant increase in total ATP. Addition of ADP in concentrations similar to those released by collagen had no effect on glycolytic activity. Addition of calcium ions produced release of nucleotides but no increase in glycolytic rate. EDTA inhibited glycolysis both in the resting platelets and in the presence of collagen. It is suggested that the stimulation of glycolysis by collagen may be due to the action of collagen on one or more rate‐limiting glycolytic enzymes.