[D-Leu1]MC-LR Has Lower PP1 Inhibitory Capability and Greater Toxic Potency than MC-LR in Animal and Plant Tissues

Two microcystins, MC-LR and [D-Leu1]MC-LR, present in La Plata Basin blooms, are differentiated by substitution of D-Alanine for D-Leucine at position 1. Our objective was to evaluate acute toxicity of [D-Leu1]MC-LR and MC-LR in mice (N:NIH Swiss) and beans (Phaseolus vulgaris). We observed variations in [D-Leu1]MC-LR lethal doses with respect to those reported for MC-LR (100 μg/kg), with an increased liver/body weight ratio and intrahepatic hemorrhages in mice exposed to 50–200 μg [D-Leu1]MC-LR/kg and slight steatosis after a single 25 μg [D-Leu1]MC-LR/kg i.p. dose. Our study in the plant model showed alterations in germination, development, morphology and TBARs levels after a single contact with the toxins during imbibition (3.5 and 15 µg/mL), those treated with [D-Leu1]MC-LR being more affected than those treated with the same concentration of MC-LR. Protein phosphatase 1 (PP1) IC50 values were 40.6 nM and 5.3 nM for [D-Leu1]MC-LR and MC-LR, respectively. However, the total phosphatase activity test in root homogenate showed 60% inhibition for [D-Leu1]MC-LR and 12% for MC-LR. In mouse liver homogenate, 50% inhibition was observed for [D-Leu1]MC-LR and 40% for MC-LR. Our findings indicate the need for further research into [D-Leu1]MC-LR toxicity since together with oxidative stress, the possible inhibition of other phosphatases could explain the differences detected in the potency of the two toxins.

The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in isolated rat adipocytes

The levels of the cytosolic serine/threonine protein phosphatases (PP) in rat adipocyte extracts have been determined, by using both reference substrates and hormone-sensitive lipase (HSL) as substrates. Adipocytes contain significant levels of both PP1 and 2A (1.6 and 2.0 m-units/ml of packed cells respectively), with lower levels of PP2C and virtually no PP2B activity. PP2A and 2C exhibit similar degrees of activity against HSL phosphorylated at site 1, together accounting for 92% of the total. In contrast, site 2 is dephosphorylated predominantly by PP2A (over 50% of total activity), whereas PP1 and PP2C contribute approx. 20% and 30% respectively to the total phosphatase activity against that site. Total phosphatase activity in the adipocyte extracts was 2-3-fold higher against site 2 than against site 1. The possible significance of these findings to the regulation of HSL activity in adipose tissue in vivo is discussed.

Activités phosphatases et carence phosphatée chez des champignons supérieurs

Six mycorrhizal and three saprophytic fungi were collected. They were cultivated in a liquid medium with or without a deficiency in phosphate. Different phosphatase activities were measured from the mycelia: phosphatase activities accessible to an external substrate, phosphatase activities in the culture medium, phosphatase activities of the whole homogenate and two parts of this homogenate (parietal and soluble). With both the mycorrhizal and saprophytic fungi, the deficiency in inorganic phosphate induced a large increase in phosphatase activities which were multiplied on the average by a factor of 72, 56, 43, 64, and 26, for the excreted, accessible, total, parietal, and soluble activities, respectively. If the relative proportions of the various activities were considered, the parietal activity, compared with the total phosphatase activity, increased in six out of nine cases under the effect of phosphate deficiency. The superiority of the parietal activity over the soluble activity was either induced by phosphate deficiency or preexisting. The accessible activity represented only a fraction of the parietal activity and this was usually smaller in a phosphate-deprived medium. Various criteria have been considered to class these fungi in terms of their response to phosphate deficiency, such as the intensity of their phosphatase response to the deficiency and the absolute values in a phosphate-deprived medium of various phosphatase activities. These various criteria do not lead to a classification entirely parallel of the different fungi and they do not discriminate between mycorrhizal and saprophytic fungi.