Giancarlo Pinzauti

Isocitrate lyase from higher plants

Abstract Work on isocitrate lyase, the first enzyme unique to the glyoxylate cycle, is reviewed.

An isocitrate lyase of higher plants: analysis and comparison of some molecular properties

A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.

Isocitrate lyase of conifers (Pinus pinea

1. Isocitrate lyase has been purified about 60 times from the conifer Pinus pinea. A first characterization was made. 2. The high instability is an important feature of this enzyme from higher plants, this causes serious problems in the purification and characterization. 3. A substantial agreement with the data from the literature was found for what concerns pH dependence of Vmax and pKm, the effect of bivalent cations and the requirement of Mg2+. 4. Kinetic studies gave evidence for a mechanism ordered uni-bi with glyoxylate being the last product released, kinetic constants were calculated, no evidence for cooperative effects was found. 5. Equilibrium constant by Haldane method calculation agrees with value calculated with isocitrate lyase from the bacterium Pseudomonas indigofera.

A new continuous optical assay for isocitrate lyase

A new continuous optical assay method for isocitrate lyase is reported. This is a coupled assay which requires lactate dehydrogenase as an ancillary enzyme. The method yields linear data up to 0.12 units/ml. The assay is also suitable for crude extracts.

Isocitrate lyase: Artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.

A continuous spectrophotometric assay for the enzymatic marker glucose 6-phosphatase

A continuous spectrophotometric assay for glucose 6-phosphatase is described. The method uses glucose dehydrogenase and mutarotase as ancillary enzymes. Glucose 6-phosphatase activity is measured by following NADH formation at 340 nm. The method is linear, at least up to 38 mU in the test which corresponds to a delta E of 0.24 min-1, when the enzyme is assayed in a microsomal fraction. We also discuss the methods suitability for subcellular fractionation. No other continuous assay for this important enzymatic marker of the endoplasmic reticulum is currently available.

Proteolytic activity in pure preparations of Pinus pinea isocitrate lyase

Abstract A proteinase was copurified with Pinus pinea isocitrate lyase. Its proteolytic action, consisting of isocitrate lyase irreversible inactivation and the appearance of enzyme forms with lower M r s, became evident when Mg 2+ was removed from pure preparations or when EDTA or SDS was added. This action was greatly reduced by Mn 2+ or oxalate, and partially by PMSF, but not by several other protease inhibitors. Some data suggest that the proteolytic activity may be adsorbed on isocitrate lyase. A model for isocitrate lyase inactivation, involving Mg 2+ dissociation, is proposed.

A New Continuous Optical Assay for Maltase and Sucrase

A new method for the assay of maltase and sucrase is reported. The method makes use of mutarotase, hexokinase and glucose 6-phosphate dehydrogenase as ancillary enzymes. The reaction is linear at least up to a delta E/min of 0.13.

Neutral maltase of human granulocytes: Localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.