Trehalose phosphorylase

Yoshida, M., Nakamura, N., and Horikoshi, K. 1995. Production and application of maltose phosphorylase and trehalose phosphorylase by strain of Plesiomonas. Oyo Toshitsu Kagaku, 42,19-25. 

Fig. 1.—Reactions Involved in the Metabolism of a,a-Trehalose. [Synthetase refers to the trehalose 6-phosphate synthetase (XDP-D-glucose D-glucose 6-phosphate l-n-glucosyl transferase), phosphatase to the trehalose 6-phosphate phosphatase, and phosphorylase to the a,a-trehalose phosphorylase.]...

It is possible that the trehalose phosphorylase described in Section V,3 (see p. 254) could be used to synthesize trehalose from n-glucosyl phosphate plus n-glucose, because the reaction appears to be freely reversible in vitro. However, as most phosphorylases appear to be degradative enzymes, this enzyme is discussed under the catabolism of trehalose (see p. 245). 

As has been described, the combined use of two phosphorylases is a powerful tool to convert one carbohydrate into another with a different structure. The idea of phosphorylase coupling was first examined by Waldmann et al. (1986), but had been employed for the synthesis of cellobiose from sucrose (Kitaoka et al., 1992), laminaribiose from sucrose (Kitaoka et al., 1993), trehalose from maltose (Yoshida et al., 1995) and kojioligosaccharides from trehalose (Chaen et al., 1999). Discovery of new phosphorylases and their application through phosphorylase coupling should be a promising area in polysaccharide and carbohydrate engineering. 

Evidence for the presence of hyperglycemic factors in the CC were reported first in P. americana (1) and supported subsequently in B. discoidalis (3). The HGHs act on the fat body, the synthetic source for trehalose in insects (22), to elevate phosphorylase activity and the conversion of glycogen stores to the precursors for trehalose synthesis (2,3). Initially, it was believed that HGHs activated phosphorylase via the synthesis of adenosine 3 5 -cyclic monophosphate (cAMP) in the same manner that glucagon or epinephrine activate liver phosphorylase in vertebrate animals (23). Injections of intact adult P. americana with synthetic Pea-CAH-I and -II result in a 50% net increase in fat body cAMP over water-injected controls accompanied by a more than 3-fold increase in fat body phosphorylase activity (24). However, the CAHs fail to elevate cAMP levels of fat bodies from P. americana in vitro even though both phosphorylase activity and trehalose synthesis increase (25). In the latter case, Ca + is essential for the action of the CAHs, and its omission from the incubation medium inhibits the hypertrehalosemic response. 

Bld-HrTH administration to B. discoidalis in vivo or to isolated fat body fails to stimulate either fat body cAMP levels or adenylate cyclase activity and supports the previous findings (25). Nevertheless, for B. discoidalis, fat body phosphorylase activity is elevated and trehalose levels increase both in vivo and in vitro, and calcium is essential in vitro in addition to Bld-HrTH. No stimulation of trehalose synthesis is noted with agents that elevate adenylate cyclase, such as forskolin, or by inhibitors of phosphodiesterase such as theophylline or isobutylmethylxanthine (IBMX). Additions of cAMP, dibutyryl cAMP or 8-bromo-cAMP are not stimulatory to trehalose synthesis either in vivo or in vitro. This same result was observed for P. americana in that neither cAMP nor dbcAMP stimulated trehalose production by fat body in vitro, and xanthine inhibitors of phosphodiesterase that should cause accumulation of intracellular cAMP were inhibitory, except for isobutylmethylxanthine (IBMX) which was stimulatory for unknown reasons (26). We have not observed a stimulatory effect by IBMX with B. discoidalis fat body in vitro. 

The exact mechanism by which the AKH/RPCHs activate fat body phosphorylase and trehalose synthesis remains uncertain. In P. americana, CC extracts with hypertrehalosemic activity neither stimulate trehalose-6-P synthase for trehalose synthesis nor increase fat body trehalose permeability (28). In all insect species tested, phosphorylase activation and glycogen degradation occur in response to CC extracts or AKH GH peptides (2.3.29-32). Therefore, all the data suggest... 

Ca + is required for phosphorylase activation in fat bodies of both P, americana (25) and discoidalis (personal observation). Addition of Ca + elevates fat body phosphorylase kinase activity in P. americana (33). and calmodulin inhibitors suppress CC-stimulated trehalose production by the fat body in vitro. However, direct addition of calmodulin to fat body phosphorylase kinase also suppresses the kinase activity. It is proposed that Ca + interacts directly with a calmodulin-like subunit of phosphorylase kinase to activate the enzyme, and the presence of exogenous calmodulin competes with the enzymic subunit for available Ca + (33). These results suggest that the HGHs may influence adipocyte Ca + levels related to phosphorylase activation to promote glycogenolysis for trehalose synthesis. Possibly, HGH-mediated fat body Ca levels may interact with polyphosphoinositides, diacyl glycerol and protein kinase C as second messengers for endocrine message transduction and phosphorylase activation. 

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