Atsumi Kamogawa

Purification and Properties of Maltose Phosphorylase from Lactobacillus brevis

Maltose phosphorylase (EC 2.4.1.8) from Lactobacillus brevis was purified 29-fold over the crude extract. The final preparation was at least 80% pure and had a specific activity of 18 units/mg protein. The molecular weights of the native enzyme and of the component dissociated in sodium dodecyl sulfate were 150,000 and 80,000, respectively. The enzyme does not contain pyridoxal-5′-phosphate as a cofactor. It can not act on maltitol, malto-triitol, sucrose, lactose and trehalose, and essentially not on isomaltose, maltobionic acid, maltotriose and maltotetraose. Inhibitory effect was observed with CuSO4, HgCl2 and p-chloromercuribenzoate. Some other properties were also examined. A possibility of using this enzyme for the analysis of maltose was proposed.

Purification and properties of glycogen phosphorylase from bovine spleen

Abstract 1. 1. The inactive form of glycogen phosphorylase (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) was purified about 180-fold in 11% yield from bovine spleen. The procedure included adsorption on starch, treatment with α-amylase and Sephadex G-200 chromatography. 2. 2. The purified enzyme revealed apparent homogeneity upon ultracentrifugation ( s 20,w = 8.5 S ) and polyacrylamide gel electrophoresis with a tendency to form active aggregates. The molecular weight determined using Sephadex G-200 was approx. 190 000 and did not change upon enzymic conversion to the active form. The spleen enzyme contained 1.03 moles of pyridoxal -5′-P/100 000 g of protein. Its amino acid composition was similar to that of rabbit muscle and pig liver phosphorylase. 3. 3. The inactive enzyme displayed activity only in the presence of AMP, and its activity increased about 4-fold upon conversion to the active form. Glucose -6-P acted as a competitive suppressor against AMP activation. Activation was also attained with cysteine, Na 2 SO 4 and NaF. 4. 4. From these results, it was assumed that spleen phosphorylase, as well as the kidney enzyme, is a type intermediate between muscle and liver phosphorylase.

Inhibition of α-glucan phosphorylase by bisulfite: Competition at the phosphate binding site

Abstract 1. 1.|NaHSO 3 was found to inhibit potato and rabbit muscle phosphorylase (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) at pH 6.0. The inhibition is completely reversible and specific for this salt. Sulfate, azide and cyanide had no effect, but bicarbonate at pH 8 showed weak inhibition. 2. 2.|The inhibition by bisulfite was competitive for glucose-1- P P 1 and arsenate. The K i values of bisulfite were determined and found to be smaller than the K m values. The interaction of the anion at the phosphate binding site occurred in an equimolecular ratio. The inhibition was more pronounced at acidic pH than at basic pH. 3. 3.|Bisulfite caused a retardation of the resolution of pyridoxal-5′- P from phosphorylase b , but not of the reductive fixation. No change in fluorescence and absorption due to the bound cofactor occurred in the reversible inhibition. The addition of hydroquinone had no effect on the rate of inhibition. 4. 4.|It is suggested from these results that the inhibition is caused by competition of bisulfite and phosphate at the phosphate binding site of phosphorylase.

Photooxidation of α-glucan phosphorylases from rabbit muscle and potato tubers

Abstract Photooxidation of α-glucan phosphorylases from rabbit muscle and potato tubers in the presence of rose bengal leads to a rapid loss of enzymatic activity which follows first-order kinetics. The process is pH dependent, being more rapid at higher pH. The inactivation is closely related to the destruction of histidine residues in the enzyme. It is suggested that histidine residues are largely responsible for the loss of enzymatic activity in the photooxidation. The inactivation of potato phosphorylase is retarded by substrates, whereas that of the muscle enzyme is not. The rate of photoinactivation of muscle phosphorylase b is increased with AMP, and decreased with ATP, ADP, IMP and glucose -6-P . This finding is considered to be closely related to the allosteric transition of phosphorylase.

Pyridoxal 5′-phosphate in α-glucan phosphorylase from the potato

1. 1. Some chemical properties of α-glucan phosphorylase (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) from potato were studied, in particular, pyridoxal 5′-phosphate bound to the enzyme protein. 2. 2. The bound pyridoxal 5′-phosphate in α-glucan phosphorylase from potato changes to a form absorbing at 390 mμ at an alkaline pH. Pyridoxal 5′-phosphate was removed from the enzyme protein by incubating in 4.2 M urea, with a concomitant loss of enzyme activity. The presence of 0.02 M NaBH4 protected the phosphorylase from inctivation by urea, but the bound pyridoxal 5′-phosphate was not attached to the enzyme under these conditions. 3. 3. The binding of pyridoxal 5′-phosphate to the enzyme protein was accomplished by reduction with 0.06 M NaBH4 at pH 6.0, or with 0.02 M NaBH4 at alkaline pH. 4. 4. The reduced enzyme protein lost its absorption peak at 330 mμ, and, in contrast to the native enzyme, showed a positive blue color in the dichloroquinone chloroimide test and a positive orange color in the p-amino acetophenone test. It was concluded that the 3-hydroxyl group of pyrdixoal 5′-phosphate was linked to the native enzyme in some way possibly by a hydrogen bond. 5. 5. In the course of reduction, decrease of absorption at 330 mμ was not found to parallel loss of enzyme activity. 6. 6. On reduction with 0.06 M NaBH4, the addition of l-lysine or Tris protected the bound pyridoxal 5′-phosphate against reductive fixation, and caused a loss of enzymic activity by some unknown mechanism.