UDP-Glucose: (1,3)-β-Glucan Synthase from Daucus carota L.

Abstract

The membrane-bound UDP-glucose-fl-(1, branes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 40C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.3Biosynthesis of plant cell wall polysaccharides is generally believed to be mediated by glycosyl transferases localized at '

Characterization of the 1,3- -glucan synthase of Aspergillus fumigatus

“…This was consistent with the property of the phosphoglucomutase . Labelling of proteins with a similar apparent molecular mass has also been detected in other plant systems (Lawson et al, 1989).…”

“…The 30 kDa family includes the 30 and 31 kDa proteins of Italian ryegrass, a 31 kDa protein from soybean and a 34 kDa protein from cotton (Meikle et al, 1991 ;Fink et al, 1990;Delmer et al, 1991). The 55 kDa family contains a doublet with molecular mass of 54 and 58 kDa in Italian ryegrass, a 57 kDa protein in both carrot and red beet, a 52 kDa protein in cotton and a 55 kDa protein from pea (Delmer et al, 1991 ;Dhugga & Ray, 1991;Frost et al, 1990;Lawson et al, 1989;Meikle et al, 1991). Two proteins labelled from A .…”

“…In fungi, it is responsible for the synthesis of the main constituent of the fungal wall and is essential for growth. Both plant and fungal enzymes are membranous and can be solubilized by detergents such as digitonin, octylglucoside or CHAPS (Fevre, 1979;Frost et al, 1992;Hayashi et al, 1987;Quigley et al, 1988;Lawson et al, 1989). No glucan synthase has been isolated until now.…”

“…The first of these probes to be used successfully was a 5-azido [32P]UDP-Glc ( 5-N3-[32P]UDPGlc; Drake et al, 1989). This compound has been used to label the catalytic subunit of plant and fungal 1,3-pglucan synthases and of the bacterial 1,4-P-glucan synthase (Frost et al, 1990(Frost et al, , 1992Lawson et al, 1989;Lin et al, 1990). More recently, K. Ng has synthesized a 5(3-p)'*'1 azido salicylamide ally1 UDP-Glc (5-'251ASA-UDP-Glc) which has been used in combination with monoclonal antibodies to identify a UDP-Glc-binding polypeptide present in the P-glucan synthase complex of Lolium mult$orurn (Meikle et al, 1991).…”

Direct Photolabeling with [32P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

“…There have been several reports of use of UDP-[3H]pyridoxal as labeled probe; in the first (22), a polypeptide of 42 kD from mung bean membranes was labeled, but labeling did not meet all the criteria expected for the catalytic subunit of callose synthase; a second report (19) indicated that a number of membrane proteins from red beet could be labeled; when the specificity of labeling was improved by a substrate protection technique, these were reduced to labeling ofpolypeptides of 200, 76, 60, and 57 kD. Wasserman's group also demonstrated the feasibility of using 5-azido-[32P]UDPglucose as an affinity labeling probe (16) and, in subsequent work using this probe (9), concluded that the 57 kD polypeptide of red beet is the most likely candidate for catalytic subunit of the enzyme based upon its enrichment upon product entrapment (see 13), its pH optimum for labeling, and its effector requirements.…”