Myeloperoxidase and eosinophil peroxidase have been isolated from outdated human blood. Peroxidase activity was extracted from washed leucocytes using 0.5 M-CaCl2 and the extract further purified by chromatography on concanavalin A--Sepharose, phenyl-Sepharose and finally by gel filtration. The final enzyme preparations were highly purified according to spectral and gel-electrophoretic criteria. Under reducing and denaturing conditions on polyacrylamide-gel electrophoresis myeloperoxidase gave rise to bands of Mr 57 000, 39 000 and 15 500, whereas the eosinophil enzyme yielded bands of Mr 50 000 and 15 500. Both enzymes were very resistant to denaturation either by the chaotropic agents urea and guanidinium chloride or by elevated temperatures. Spectral properties of the native and reduced forms of the enzymes are reported.

Synthesis and processing of myeloperoxidase were examined in metabolically labeled cells of the human promyelocyte line HL‐60 and in an in vitro rabbit reticulocyte lysate system directed with HL‐60 mRNA. Radioactivity labeled products were isolated by immunoprecipitation and analyzed by gel electrophoresis and fluorography. In vivo, myeloperoxidase was labeled initially as a 85‐K glycosylated polypeptide (75 K after treatment with endo‐beta‐N‐acetylglucosaminidase H). This polypeptide was soon processed to an 81‐K intermediate and to smaller mature fragments of 60 K and 13 K within approximately 1 day. A minor portion of the precursor was converted to fragments of 40 K and 43 K. The pattern of labeled polypeptides of mature myeloperoxidase was similar to that of the enzyme purified from human leucocytes. The modifications of the polypeptide and of the oligosaccharide side chains in myeloperoxidase resembled those known to occur during the processing of lysosomal enzymes. In the absence or presence of dog pancreas membranes, myeloperoxidase was synthesized in vitro as a 76‐K polypeptide or a 87‐K glycosylated polypeptide, respectively. In HL‐60 cells [32P]phosphate was incorporated into endo‐beta‐N‐acetylglucosaminidase H‐sensitive oligosaccharides. The presence of phosphorylated oligosaccharides was inferred from the fact that endocytosis of leucocyte myeloperoxidase in fibroblasts was sensitive to mannose 6‐phosphate. It is suggested that myeloperoxidase is synthesized in the rough endoplasmic reticulum as a precursor of larger molecular mass and that the oligosaccharide side chains in the precursor are modified to contain mannose 6‐phosphate residues which may be involved in the segregation and transport of the precursor.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

SUMMARY Intraventricular infusions of octopamine which raised brain octopamine concentrations more than 20 000-fold resulted in reductions in brain noradrenaline and dopamine by as much as 90% without affecting the alertness or activity of normal rats. As this reduction of brain catecholamines is much greater than any reported in hepatic coma, we do not believe that values observed in experimental hepatic failure have aetiological significance for the encephalopathy that ensues.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.