Masanao Kobayashi

Multiple molecular forms of phosphoprotein phosphatase: III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?

Multiple molecular forms of phosphoprotein phosphatase II. Dissociation and activation of phosphorylase phosphatase from rabbit skeletal muscle

Abstract 1. 1.|The phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) activity that was found in the preparation of phosphoprotein phosphatase from rabbit skeletal muscle was resolved into two fractions by gel filtration (Sephadex G-150) or sucrose density gradient centrifugation. These two fractions are similar to those of the phosphohistone phosphatase and glycogen synthetase-D phosphatase activities (Kato, K. and Sato, S. (1974) Biochim. Biophys. Acta 358, 299–307). The apparent K m values for phosphorylase a of the larger form ( s 20, w = 6.7) and the smaller form ( s 20, w = 3.8) were 13.3 μM and 6.7 μM (based on the 32 P content in [ 32 P]phosphorylase a ), respectively. No metal-ion requirement or stimulation was observed in both forms. 2. 2.|The larger form of phosphorylase phosphatase was dissociated by freezing, incubation with mercaptoethanol or dithiothreitol, or trypsin digestion into a molecular size similar to that of the smaller form as was phosphohistone phosphatase. Dissociation of the larger form of phosphorylase phosphatase resulted in a several-fold increase of activity with the same K m (13.3 μM) for phosphorylase a . 3. 3.|The crude extract (20 000 × g , 30 min) of rat skeletal muscle was found to have the larger and smaller forms of both phosphorylase phosphatase and phosphohistone phosphatase.

Isolation of a substance toxic to mitochondrial function from the burned skin of rats

Abstract Extract from burned skins of rats contained an activity impairing the respiratory control index (RCI) of liver mitochondria in vitro . This mitochondria-toxic activity was significantly enhanced 1–2 weeks after the thermal injury. When analysed 2 weeks post burn, most of the toxic activity appeared to be associated with the eschar. The activity was heat stable and was easily extracted with ether from the acidified aqueous extract. When tested with this ether extracted preparation, not only RCI but ADP/O and the rate of ATP-formation were also impaired in a dose-dependent manner. At higher concentrations of the ether extract, electron transport itself was inhibited. Thin layer chromatography of the above preparation revealed 5 iodine-positive bands; the toxic activity was associated with only one of them (Band 3). This substance could be purified by high-performance liquid chromatography essentially to a single band on thin layer chromatography. The extract from sham-operated normal skins also contained this substance but much less in amount—2–20 per cent of that in burned skins when estimated by absorbance at 215 nm. Current analyses indicate that the mitochondria-toxic substance in burned skins may be a lipid, probably a derivative of fatty acids, which is a normal component of the skin, and may be produced in excess after thermal injury.