Glucan synthase

The glucan synthase inhibitor caspofungin (intravenous formulation) is new on the market for the treatment of invasive aspergillosis in patients whose disease is refractory to, or who are intolerant of, other therapies. During the clinical trials fever, infused vein complications, nausea, vomiting and in combination with cyclosporin mild transient hepatic side effects were observed. Interaction with tacrolismius and with potential inducer or mixed inducer/inhibitors of drug clearance was also seen. 

Saugy, M., Farkas, V., and Maclachlan, G. (1988) Phosphatases and phosphodiesterases interfere with 1,3-b-D-glucan synthase activity in pea epicotyl membrane preparations. EurJ.Biochem. 177 135-138. 

The echinocandins have a unique target for their antifungal activity—specifically, (5-1,3-glucan synthase, an enzyme that produces an important component of the fungal wall. Caspofungin is currently the only product of this class that is FDA approved it is indicated for treatment of IA refractory... 

Abbreviations. a-M, a-mannosidase AP, acid phosphatase as-ni-ATPase, anion-stimulated, nitrate-inhibitable ATPase CCR, NAD(P)H-dependent cytochrome oreduc-tase cs-vi-ATPase, cation-stimulated, vanadate-inhibitable ATPase, CAT, catalase GS 1/11, glucan synthase 1 or 11 IDPase, inosine diphosphatase cs-PPase, cation-stimulated pyrophosphatase RNA polymerase, DNA-dependent RNA polymerase TP-25, 25 kDa tonoplast integral protein. 

Kause HL, Jeblich W. Synergistic activation of l,3-(J-D-glucan synthase by Ca2+ acid polyomers. Plant Sci 1986 43 103-107. 

Kobayashi S, Koga K, Hayashida O, Nakano Y and Hasegawa Y (1990) Specific inhibition of insoluble glucan synthase (GTF-1) by Maillard reaction products from casein and albumins. Agric Biol Chem 54, 1417-1424. 

Castelli MV, Cortes JCG, Escalante AM, Bah M, Pereda-Miranda R, Ribas JC, Zacchrno S A (2002) Inhibition of (1,3)-P-Glucan Synthase by Glycoresins from Convolvulaceous Plants. Planta Med 68 739... 

PF had been proposed as the terminal complex (23) and associated pores were reported on the outer membrane EF (24). Due to their proximity to the site of cellulose ribbon extrusion from the cell surface, these structures were assumed to be responsible for cellulose synthesis. A model was advanced in which cellulose synthase was localized on the outer membrane, which invoked adhesion sites between the outer and plasma membranes as a mechanism to explain the transfer of uridine-diphosphoryl-glucose (UDPG) from the cytoplasm to the cellulose synthases (25,26). However, when the outer and plasma membranes of Acetobacter were isolated separately by density-gradient centrifugation, the cellulose synthase activity was localized only in the plasma membrane fraction (27). Therefore, the linear structures observed on the Acetobacter outer membrane, while they may be associated in some manner with cellulose biosynthesis, are probably not the cellulose synthase terminal complexes. Since no ultrastructural evidence for adhesion sites between the outer and plasma membranes has been presented, a thorough investigation of the mechanism of / (1-4) glucan chain translocation from the cytoplasmic membrane to the outer membrane in Acetobacter xylinvm is now in order. 

Tools have recently been developed that should enable identification of the catalytic subunits of the plasma membrane / -(l,3)-glucan synthase from red beet storage tissue and other plant sources. These include an efficient procedure for solubilization and enrichment of this enzyme using the detergent CHAPS, generation of antibodies, and use of affinity labels such as UDP-pyridoxal. 

Preparation of Glucan Synthase Fractions. Microsomal and plasma membranes were isolated by differential and density-gradient centrifugation. CHAPS-solubilized glucan synthase (CSGS) was prepared by the two-step procedure (4,9). In step 1, contaminating proteins were extracted with... 

Figure 1. Electrophoretic profiles ofglucan synthase fractions purified from red beet. Proteins were transferred to nitrocellulose and stained by colloidal gold followed by silver overlay. Lane 1, solubilized enzyme (CSGS) Lane 2, reconstituted glucan synthase (RCGS).

Table I. Immunoprecipitation of CHAPS-Solubilized Glucan Synthase 1...

Affinity Labeling with UDP-pyridoxal. Read and Delmer (21) utilized the substrate analog UDP-pyridoxal to inhibit mung bean glucan synthase. This affinity label inhibited glucan synthase at micromolar levels and inhibition was protected against with UDP-glucose. A 42 kD polypeptide could be labelled with [3H]UDP-pyridoxal. 

Table II shows that UDP-pyridoxal had a similar inhibitory effect on red beet glucan synthase. It inhibited activity at much lower concentrations than other covalent modification reagents, such as N-ethylmaleimide (cysteine), phenylglyoxal (arginine) and formaldehyde (lysine). UDP-pyridoxal had an I50 that is 62-fold lower than formaldehyde.

Table II. Chemical Modification of CHAPS Solubilized Glucan Synthase...

Table III. Inhibition of Plasma Membrane Glucan Synthase by UDP-pyridoxal...

Figure 2. Electrophoretic profiles of glucan synthase fractions purified from celery. Proteins were transferred to nitrocellulose and stained by colloidal gold. Symbols PM, plasma membranes SOL, CHAPS-solubilized RC, reconstituted glucan synthase preparations. Plasma membranes were isolated by two-phase partitioning. Specific activities of the three membrane preparations were 398, 1355, and 626 nmol/min/mg, respectively. The low specific activity of RC relative to SOL may be a reflection of enzyme instability following the gel filtration step.

In vivo linear a-l,4-glucans are synthesized from ADP-glucose by the enzyme glycogen synthase [94-97]. The enzyme, as well as the monomer, are quite sensitive and therefore most researchers (at least in the field of polymer science) prefer to use phosphorylase for the synthesis of amylose. 

A characteristic feature of the SuSy isoforms is a conserved phosphorylated serine residue near the N-terminus [8-10]. In-vivo studies have demonstrated that phosphorylation and dephosphorylation direct the distribution of SuSy isoforms in the plant cell [10-12]. The soluble phosphorylated SuSy interacts with the actin cytoskeleton in the cytoplasm [13], and the dephosphorylated SuSy isoforms are targeted to the cell membrane to form complexes with other enzymes, e.g., glucan synthase, catalyzing cellulose biosynthesis from sucrose [4, 10, 14]. In this respect, recent studies on the dephosphorylated enzymes by cloning and expression of SMS genes in E. coli have shown differences in some biochemical features when compared to the enzymes isolated from the corresponding plant material. Recom-... 

Caspofungin Blocks 3-glucan synthase Prevents synthesis of fungal cell wall Fungicidal Candida sp also used in aspergillosis IV only duration, 11-15 h Toxicity Minor gastrointestinal effects, flushing Interactions Increases cyclosporine levels (avoid combination)... 

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