On the mechanism of the inhibition of glutamine synthetase and creatine phosphokinase by methionine sulfoxide.

Beta amyloid peptides (A beta), etiologically associated with Alzheimer's disease (AD), have been shown to inhibit both glutamine synthetase (GS) and creatine phosphokinase (CPK) in vitro. These two enzymes are affected in AD and are sensitive to oxidative stress. Residue 35 of the A beta 25-35, the most potent section of the 40-42 amino acid long fragment of amyloid precursor protein (APP), is a methionine, which has been reported to be oxidized to methionine sulfoxide presumably via a free radical oxidation process. We questioned whether methionine sulfoxide would inhibit GS and CPK directly and if this inhibition also involved free radical oxidative stress. In this report, we demonstrate that methionine sulfoxide inhibits GS by about 50% and CPK by about 25% at 20 mM concentration. Neither intact SOD, nor ascorbate inhibit the action of methionine sulfoxide completely, with regard to the inactivation of GS. These results indicate that the action of methionine sulfoxide may not be directly due to the oxidation of GS by free radicals. In fact, the presence of exogenous proteins, such as denatured SOD or catalase, inhibit the action of methionine sulfoxide as, or more effectively than, the addition of active free radical antioxidant enzymes.

Subtractive antibody to a human immunosuppressive lymphokine affinity isolates a suppressive factor and blocks its function.

Hybridoma suppressor factor(s) (HSF), secreted by a human thymus hybridoma (8E-24) established in this laboratory, suppresses Ig as well as IL-2 synthesis by peripheral blood mononuclear cells (PBMC). To aid in the characterization of this lymphokine, we prepared a subtractive antibody to HSF using the products of the hybridoma parent cell line to generate antibodies to irrelevant proteins. The concentrated supernatant fluid of the hybridoma parent cell line (CEM) was used to generate rabbit antibodies and titers of anti-CEM were monitored by enzyme immunoassay (EIA). Next, to remove factors shared by both the parent cell line and hybridoma, the concentrated supernatant fluid of 8E-24 (crude HSF) was passed over an immunoaffinity column, composed of protein A beads coupled to anti-CEM. The 'subtracted' HSF, termed partially purified HSF, was shown to be suppressive in vitro and was then used to prepare a second rabbit antisera. Using partially purified HSF as antigen, the presence of specific antibody was monitored by EIA. This antibody (anti-HSF) was used to prepare another immunoaffinity column by covalently coupling this antibody to protein A beads. Factors bound and then eluted from this affinity column were shown to inhibit IL-2 production by PBMC in a manner similar to HSF. Specific activity of the affinity purified HSF was 50 times that of partially purified HSF. Furthermore, the suppressive activity of affinity purified HSF was abrogated in the presence of anti-HSF. Western blot analysis performed on the concentrated crude HSF, using both the anti-HSF and anti-CEM antibodies, revealed the presence of several bands that selectively reacted with anti-HSF and not anti-CEM. Each of these bands were present in the affinity purified HSF. Of particular interest due to their similar size to the suppressive agent are a band at 12 kDa that reacts selectively with anti-HSF and is detected in crude and affinity purified HSF and a band at 10 kDa. In summary, this protocol resulted in the detection and separation of hybridoma specific proteins within the predicted size range of the suppressive lymphokine.

Stimulation of prostacyclin synthesis in rats treated with phenobarbital.

The effect of phenobarbital treatment on the synthesis of prostacyclin in coronary vascular microsomes was studied in Sprague Dawley rats. It was found that the treatment increased the synthesis of prostacyclin by nearly 100%. The treatment also resulted in an increase in HDL and a decrease in LDL in the serum. In vitro effects of HDL and LDL on the microsomal synthesis of prostacyclin showed that the synthesis was stimulated by HDL and inhibited by LDL. Hence it appears that the increase in prostacyclin synthesis resulting from phenobarbital treatment was at least partly due to increased level of HDL and decreased level of LDL in the serum.

Inhibition of thromboxane A2 synthesis in rats treated with phenobarbital.

The effect of phenobarbital administration on serum lipoproteins and thromboxane A2 synthesis in platelets was studied in rats. Phenobarbital decreased the serum LDL level by 33% and increased the HDL level by more than 15%. The synthesis of thromboxane A2 in the platelets of the phenobarbital treated animals was found to be reduced by 43%. Thromboxane A2 synthesis in the platelets of the control animals was inhibited by HDL and stimulated by LDL. Hence it appears that the decreased thromboxane A2 synthesis in the platelets of phenobarbital treated rats was at least partly due to the increased HDL and decreased LDL in the serum. Phenobarbital treatment also caused a 15% increase in the serum HDL-cholesterol although it did not have any significant effect on the total serum cholesterol.