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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.

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 method's suitability for subcellular fractionation. No other continuous assay for this important enzymatic marker of the endoplasmic reticulum is currently available.

Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.

Phosphate esters of vitamins D2 and D3 (D2-O-P and D3-O-P) are hydrolyzed by the alkaline phosphatase from calf intestine (CAPase). pH optimum and Km values are in agreement with those reported for other substrates for intestinal alkaline phosphatase. No effect of activation was noted at low concentrations of D2-O-P and D3-O-P (10(-5)-10(-4)M) in contrast with that stated in earlier works. At higher concentrations (1-4 mM) of the phosphorilated vitamins, accordingly with their function as substrates of CAPase, an inhibitory effect on the hydrolysis of p-nitrophenylphosphate was observed.

Neutral maltase from human granulocytes has a different substrate specificity from the human neutral maltase of kidney, though it has been reported that these two enzymes are immunologically similar. We report here that human granulocyte neutral maltase is similar to the neutral maltase from rat's kidney as regards the substrate specificity and the inhibition by Tris and maltodextrins. We also report a different thermal stability that might imply some structural differences between the two enzymes.

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 is a key catalyst of the glyoxylate cycle. A feature of the enzyme from higher plants is the high instability, that causes innumerable problems in working for characterization of the enzyme. The present communication demonstrates that the optimal conditions for the storage of isocitrate lyase from Pinus pinea are: the use of a low temperature (possibly below -20 degrees C), the realization of a high endogenous protein concentration of the enzyme preparations, or, above all when long storage conservation is necessary, the preservation of the enzyme in dried form (acetone precipitation), under vacuum at 4 degrees C. The data reported in this paper seem to exclude, in the above studied conditions, a role for serine proteases in the destabilization of the enzyme. The thiol compounds are not determinant and no effect is obtained by adding exogenous proteins (serum albumin, beta-fructosidase).

A neutral maltase immunologically similar to this of kidney exist in human granulocytes. We have studied some kinetic properties of this enzyme on a microsomal fraction of granulocytes. Its optimal pH is very closed of 6.8 and this enzyme, highly specific for maltose, hydrolysis very weakly the nigeriosis. Maltotriose, maltotetraose and maltopentanose are inhibitors of this enzyme, which is not inhibited by all disaccharides studied.

We found that the Mg-isocitrate complex is the true substrate for pine isocitrate lyase and that magnesium acts as a non-essential activator. Both the non-activated and the activated enzyme forms are catalytically active. Our model is consistent with the presence of two Mg-binding sites with different affinities: an activator site with high affinity in addition to the catalytic site with lower affinity. This may result in a complex, fine regulation of isocitrate lyase activity by magnesium. The affinity of the free enzyme for isocitrate is very low. Moreover, free isocitrate does not bind to the activated enzyme, nor it can yield a catalytically active form by binding to an enzyme species whose catalytic site has already been bound by magnesium.

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.