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  • Author: Hiroyuki Kodama x
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Abstract

The effect of various amino acids and MnCl2 on prolinase activity in erythrocyte lysates from a healthy individual and a patient with prolidase deficiency was investigated. A concentration of 0.1 mM MnCl2 increased prolinase activity in normal erythrocytes against pro-gly, pro-glu, pro-leu, pro-ser and pro-phe, but inhibited that against pro-ala, pro-val, pro-met and pro-asp. However, prolinase activity against these iminodipeptides was enhanced by pre-incubation with glycine, independent of MnCl2. The same studies on erythrocytes from a prolidase-deficient patient showed almost the same results as the normal control, except that prolinase activity against pro-gly and pro-ser was slightly inhibited by adding 0.1 mM MnCl2. Some amino acids, glutamic acid and glutamine, slightly enhanced prolinase activity against progly in erythrocytes from both the normal control and the prolidase-deficient patient, but N-acetyl-L-glutamic acid, γ-aminobutyric acid (GABA) and β-alanine showed no effect. Branched amino acids, L-valine, L-leucine and L-isoleucine strongly inhibited the prolinase activity against pro-gly. However, conversely, their isomers, D-valine, D-leucine and D-isoleucine, enhanced it.

The kinetics of prolinase activity in the erythrocytes from both the normal individual and the prolidase-deficient patient were also studied. Their Km values were changed by adding glycine or 0.1 mM MnCl2, but Vmax values were almost the same.

Abstract

We investigated the effects of S-(1,2-dicarboxyethyl) glutathione (DCEG) and S-(1,2-dicarboxyethyl) cysteine (DCEC) on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils.

When the cells were preincubated with DCEG, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was enhanced in concentration-dependent manner but DCEC showed no effect. The influence of DCEG and DCEC on phorbol 12-myristate 13-acetate-induced superoxide generation showed no effect. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by DCEG and DCEC in concentration-dependent manner. The suppression of DCEG was more effective than that of DCEC.

The superoxide generation induced by fMLP in the DCEG-treated cells was suppressed by genistein. DCEG enhanced tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins in human neutrophils. The tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins was suppressed by genistein. The enhancement of tyrosyl phosphorylation of these proteins in the DCEG-treated cells was parallel to the enhancement of the fMLP-induced superoxide generation.

Abstract

Prolidases I and II were highly purified from human erythrocytes. The effects of various amino acids, MnCl2 and mercaptoethanol, on these two enzymes were investigated. Normal prolidase II was very labile in the absence of MnCl2 or mercaptoethanol. The activity of prolidase II was maintained at about 76% by preincubation with MnCl2; it was then activated up to 140% by treatment with mercaptoethanol for 60 minutes at 37 °C. Normal prolidases I and II showed the highest activity against glycylproline or methionylproline in the presence of MnCl2. The activity of prolidase I against glycylproline was enhanced strongly by glycine and MnCl2, but not activated in the absence of MnCl2. The activity of prolidase II against methionylproline was enhanced three-fold in the presence of glycine and MnCl2, but its activity against glycylproline was very low even in the presence of MnCl2. A stronger enhancement of this activity was found in normal erythrocytes, and a lower level of this activity was found in erythrocytes of patients treated with glycine, MnCl2 and mercaptoethanol compared to those treated with glycine and MnCl2. The activity of prolidase II against methionylproline in all erythrocytes, of normal humans and of patients, was strongly activated by the addition of glycine with MnCl2 but suppressed by the addition of mercaptoethanol. Clin Chem Lab Med 2003; 41(10):13231328