Purification and properties of coproporphyrinogen oxidase from the yeast Saccharomyces cerevisiae.

Coproporphyrinogen oxidase has been located in the cytosol of yeast cells. The enzyme was purified to homogeneity from a heme mutant strain exhibiting a high specific activity (15-20 enzyme units/mg soluble protein compared to 1-2 enzyme units/mg soluble protein of by the wild-type strain). The final preparation was homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Mr = 35,000) and isoelectrofocusing (pI = 6.2). Gel filtration on AcA 44 gave a relative molecular mass of 70,000. N-terminal amino-acid sequence analysis revealed a single polypeptide chain. Thus the enzyme appears to be a dimer with identical subunits. Two iron atoms/molecule of native protein were detected; they could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. However the involvement of the iron atoms in the oxidative catalytic activity of the enzyme was not demonstrated. The Km value for coproporphyrinogen was 0.05 microM. The enzyme was active only when molecular oxygen was used as electron acceptor; no anaerobic activity could be detected. Thiol-directed reagents partially inhibited the enzyme, indicating that an SH group is required for activity. Yeast coproporphyrinogen oxidase was activated by phospholipids or neutral detergents as described for the bovine liver enzyme.

Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase.

We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins). Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast. These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C. Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium. The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M. Camadro, D. Urban-Grimal, and P. Labbe, 1982, Biochem. Biophys. Res. Commun. 106, 724-730). Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase. The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested. However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity. This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml. Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min.

Total pancreatectomy in the treatment of acute necrotising and hemorrhagic pancreatitis. Indications-technique.

The place of total pancreatectomy in the treatment of pancreatitis is still not clear: the author is in favour of this operation and gives the indications, surgical technique, complications and results. The operation is indicated in cases of necrosis involving more than 2/3rds of the whole of the head and part of the body of the pancreas. The duodenum and pancreas should be removed in one piece and intestinal continuity should be restored performing choledocho-jejunal and gastro-jejunal anastomoses. It is important to carry out this operation early, between the 3rd and 6th days, treating all areas of necrosis before the lesions become the site of uncontrollable infection. Seven patients out of 9 are still alive, on the 25th of July 1975; they all have easily controlled diabetes, a low fat diet and are receiving pancreatic extract. We have recently operated a 10th case, and the patient is alive 2 months later.

Some properties of yeast mitochondria prepared by a rapid mechanical procedure.

A method is described for preparing yeast mitochondria rapidly (within one hour) by using the MSK Bronwill Cell homogenizer. Yeast mitochondria obtained by the method exhibit relatively good respiratory controls and ADP/O ratios. The method is convenient for small or large amounts of yeast cells (from 5 to a hundred grams, wet weight) and gave a yield of 3 to 5 mg protein/g wet weight of yeast mitochondria.

[Total pancreatectomy in acute necrotizing hemorrhagic pancreatitis. Indications and technic]. / La pancréatectomie totale dans la pancréatite aiguë nécrosante et hémorragique. Indications. Technique

The place of total pancreatectomy in the treatment of pancreatitis is still ill- defined. The author makes a plea for this operation and notes the indications, the surgical technique and its results and possible complications. The operation is indicated in cases of total or 2/3 rds necrosis of the gland, in cases involving the head of the pancreas and part of the body. The gland should be dissected out and continuity should be restored by choledoco-jejunal and gastro-jejunal anastomoses. The important thing is to carry out this operation early, between the 3rd and 6th day, treating the areas of necrosis before the lesions become the site of uncontrolled infection. 7 patients out of 9 operated on in this way, are alive with easily controlled diabetes, a low fat diet and pancreatic extract.