Alkaline phosphatase of Drosophila melanogaster. II. Biochemical comparison among four allelic forms

Biochemical analyses of partially purified preparations of APH-4 and -6 (common allelic forms) and APH-2 and -10 (rare allelic forms) of D. melanogaster reveal that the two common forms are similar in all properties investigated except for pH optimum (8.0 vs. 8.5). The common and rare forms share certain properties in common but differ in that the common forms are more stable to heat and more sensitive to inhibition by inorganic phosphate. With respect to such properties as substrate preferences and K _i values for inorganic phosphate, the common forms and APH-2 are similar to one another, whereas APH-10 is distinctly different. All four activities show preference for a phosphoaromatic compound as substrate, with O-phosphotyrosine being the best substrate of biological origin. Transphosphorylation, as related to these allelic forms of APH, is discussed.Paper No. 3892 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. This study was supported by Atomic Energy Commission Contract AT-(40-1-)-3980.     

The reversal of phenylarsenoxide inhibition of keto acid oxidation in mitochondrial and bacterial suspensions by lipoic acid and other disulphides

1. Inhibition of pyruvate oxidation in suspensions of Aerobacter aerogenes cells and of isolated mitochondria from rat heart and liver by phenylarsenoxide is prevented by an excess of lipoic acid, whereas inhibition due to certain bivalent cations is not. 2. In both systems inhibition persists when the bacteria and mitochondria are recovered and resuspended in fresh media in the absence of the inhibitor. Persistent inhibition due to preincubation with phenylarsenoxide, but not with the metal ions, is reversed by lipoic acid and by certain other disulphides. 3. 2,3-Dimercaptopropan-1-ol prevents the inhibition of pyruvate oxidation by phenylarsenoxide and by bivalent cations in both mitochondria and bacterial cells. 4. In aerobic suspensions of mitochondria and bacteria disulphides such as lipoic acid are reduced rapidly to dithiols. Reduction is inhibited by Co(2+), Ni(2+), Cd(2+) and Zn(2+), but not by phenylarsenoxide. 5. It is concluded that the inability of lipoic acid to prevent the action of the metal ions on pyruvate oxidation is due to the inhibition of its reduction to the effective dithiol.     

Structural Changes in Mammalian Cells Associated with Cooling to -79°C

Suspensions of HeLa and S37 cells, with and without added glycerol, were cooled in stages to -79°C. and held at that temperature for 30 minutes. After warming to room temperature the cells were fixed, sectioned, and compared by phase contrast and electron microscopy with similar specimens kept at room temperature. Correlated viability tests were made. Abnormal cytological characteristics, visible with the phase contrast microscope, were clearly related to the sequence of freezing and thawing, and the proportion of altered cells was highest in specimens cooled without glycerol. Electron microscopy showed that even in the presence of glycerol all cells were markedly altered, with distinctive vesiculation and disruption of the various intracellular membranes. There is evidence that much cytoplasmic damage is compatible with survival, but it seems likely that separation of the two layers of the nuclear envelope and rearrangement of the nuclear contents are signs of irreversible damage. The findings lend some support to the belief that cell death on cooling is due largely to denaturation of semipermeable membranes, caused by the increasing concentration of electrolytes.