Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Inhibition of the yeast respiratory system by Zn-protoporphyrin and effect of photolysis of this substance]

We have shown earlier that yeast cells grown in synthetic mediums supplemented with Zn++ accumulate large amounts of Zn-protoporphyrin within their mitochondria. This accumulation is accompanied by an inhibition of respiration (3). This study deals with the effect of light on the respiratory inhibition and the release of respiratory control which are observed if Zn-protoporphyrin is added to isolated mitochondria which are initially devoid of this pigment. In addition, we have studied the effect of light on the respiratory inhibition exerted by Zn-protoporphyrin accumulated in vivo. The following results were obtained: 1) The light-induced destruction of Zn-protoporphrin which had been added in vitro to Zn-protoporphyrin-free mitochondria significantly inhibits respiration and phosphorylation. Under these conditions, the extent of the inhibitions increases with the concentration of the added Zn-protoporphyrin and the duration of illumination. 2) Accumulation of Zn-protoporphyrin within the cells causes an inhibition of the respiratory activities and the activities of succinate-cytochrome c reductase and NADH-cytochrome c reductase of the mitochondria. Illumination of the isolated mitochondria from Zn-protoporphyrin-containing cells enhances the inhibition of these activities. No light-induced inhibition of these activities is observed with mitochondria from cells devoid of Zn-protoporphyrin.     

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8 kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.     

Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding

Background

Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.

Methods

To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.

Results

We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.

General significance

Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.