The presence of an active transport system for glucose-1-phosphate in Agrobacterium tumefaciens was demonstrated from the following observations. (i) The bacterium could grow on a medium containing glucose-1-phosphate as carbon source; (ii) the entry of glucose-1-phosphate into the resting cells occurred against concentration gradient obeying Michaelis-Menten kinetics; and (iii) the entry reaction was energy-dependent. The transport system for glucose-1-phosphate was formed inducibly by growing the organism on a glucose-1-phosphate or sucrose medium. From the inhibition and kinetics studies it was found that the transport system had a high specificity for glucose-1-phosphate with a high affinity, K(m) value of 4.5 x 10(-6)m at pH 8.2. The existence of glucose-1-phosphate binding factor was proved in the shock fluid which was prepared from the cells grown on both glucose-1-phosphate and sucrose media by osmotic shock.

Biological Transport, Active↗

PubMed1970

Alpha-3-ketoglucosidase of Agrobacterium tumefaciens.

A 3-ketosucrose-degrading enzyme was purified 80-fold from the sonic extracts of Agrobacterium tumefaciens IAM 1525 grown on a sucrose-containing medium. The enzyme catalyzes hydrolysis of alpha-3-ketoglucosides such as 3-ketosucrose, 3-ketotrehalose, 3-ketomaltose, and 3-ketoglucose-1-phosphate but not of beta-3-ketoglucosides, beta-3-ketogalactosides, and other glycosides such as sucrose, trehalose, maltose, glucose-1-phosphate, cellobiose, lactose, or raffinose. From the strict substrate specificity of this enzyme, the name alpha-d-3-ketoglucoside 3-ketoglucohydrolase (trivial name, alpha-3-ketoglucosidase) was proposed. K(m) values for 3-ketosucrose and 3-ketotrehalose were 3.9 x 10(-3)m and 4.8 x 10(-3)m, respectively. Optimum pH was 8.0 to 8.3. 3-Ketoglucose, a reaction product from alpha-3-ketoglucosides by the enzyme, behaved as a strong inhibitor. Physiological significance of this enzyme in the disaccharide metabolism of this bacterium was discussed.

Carbon Isotopes↗

PubMed1970

Presence of binding site for alpha-amylase and of masking protein for this site on mycelial cell wall of Aspergillus oryzae.

Mycelial cell wall of Aspergillus oryzae M-13 grown in an alpha-amylase-forming medium could not bind alpha-amylase (Taka-amylase A, EC 3.2.1.1). However, by treatment with 1.0 n NaOH at 100 C for 30 min, the wall gained the ability to bind alpha-amylase. This phenomenon was caused by removal of a factor (designated as masking factor) which masked the binding site for alpha-amylase. The masking factor was purified as a preparation giving a single peak in both ultracentrifugation (1.6S) and by gel electrophoresis (M(BPB), 1.0). Approximately 20 mug of the purified factor, bound to 10 mg of the alkali-treated mycelial cell wall, prevented the binding of approximately 100 mug of alpha-amylase or released approximately 100 mug of alpha-amylase which previously was bound to the alkali-treated wall. These findings indicate that the factor has much higher affinity than alpha-amylase for the binding site on the mycelial wall. The masking factor was inducibly formed accompanying the secretion of alpha-amylase.

Amylases↗

PubMed1970