Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)

S.S Ghosh,J Adhikari,Barasat Government College, West Bengal, India ,Hiralal Mazumdar Memorial College For Women, Kolkata, India ,S Basu,M Das

doi:10.15407/ukrbotj77.05.386

Abstract

The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.

Highlights

Aquatic ferns bear specialized and unique reproductive structures known as ‘sporocarps’
D-fructose-1,6-bisphosphate, D-fructose6-phosphate, D-glucose-6-phosphate, D-galactose-6-phosphate (Gal-6-P, disodium salt), D-fructose-6-phosphate (F-6-P, di-sodium salt), D-mannose-6-phosphate (M-6-P, di-sodium salt), bovine serum albumin (BSA), were obtained from SRL (Mumbai, India). 2-Mercaptoethanol (ME), ammonium molybdate, acetic acid, ammonium sulphate, ammonium chloride, copper chloride, potassium chloride, tris(hydroxymethyl)aminomethane, trichloroacetic acid (TCA), dipotassium hydrogen phosphate, magnesium chloride, hydrochloric acid, sodium chloride, sulphuric acid, zinc chloride were purchased from E Merck India Ltd. (Mumbai, India). 2-Glycerophosphate was obtained from BDH, England
In the young sporocarps of all the experimental samples, the specific activity of FBPase was relatively lower than the mature sporocarps obtained from the same source (Table 1)

Summary

Introduction

Aquatic ferns bear specialized and unique reproductive structures known as ‘sporocarps’. The young immature sporocarps are green, leathery and photosynthetic, while on maturation they become non-photosynthetic, blackish brown in colour and eventually break down as a result of disintegration of the indusial and the sporangial walls releasing the spores to the external environment. These unique desiccation tolerant structures have been extensively studied from various angles like morphology, anatomy, Український ботанічний журнал, 2020, 77(5). It has been reported earlier that adequate glucose concentration is the central parameter in living organisms for optimization and fulfilment of all metabolic events either directly or indirectly (Eckstein et al, 2012) In plants this sugar is available in all parts basically via photosynthesis in leaves followed by phloem translocation. The conversion of immature fern sporocarps into mature and senescent ones involves a definite loss of photosynthetic activity which in turn indicates the possible occurrence of gluconeogenic pathway and the concomitant involvement of the gluconeogenic marker enzyme, fructose-1,6bisphosphatase (FBPase)

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Discussion

Conclusion