Johannes Gunawan

Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Abstract: An enzymic activity of rat brain that liberates radioactive free aldehydes from 1‐[1‐14C]alk‐1′‐enyl‐sn‐glycero‐3‐phosphoethanolamine (lysoplasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21‐day‐old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg2+, Ca2+) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone‐dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1‐[1‐14C]alk‐1′‐enyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.

Alkenylhydrolase: a microsomal enzyme activity in rat brain

Abstract: Rat brain microsomes have the capacity to liberate radioactive free aldehydes from 1‐[1‐14C]alk‐1′‐enyl‐sn‐glycero‐3‐phosphoethanolamine (lysoplasmalogen). Glycerophosphoethanolamine was found using 1‐alk‐1′‐enyl‐sn‐glycero‐3‐phospho‐[3H]ethanolamine. The ratio of both products released by lysoplasmalogenase action was 1:1. Another enzymic activity could be demonstrated, which hydrolyzes lysoplasmalogen at the hydrophilic part of the molecule, a lysophospholipid phospho diesterase. Thus, 1‐[1‐14C]alk‐1′‐enylglycerol was detected as well as [3H]ethanolamine, again in a molar ratio, from the respective labeled substrates. This enzyme possesses nearly the same affinity toward the substrate as lysoplasmalogenase. Whereas the lysophospholipid phos phodiesterase is totally inhibited in the presence of NaF or EDTA, lysoplasmalogenase activity is not affected by these reagents. 1‐[1‐‘14C]Alk‐l’‐enylglycerol acts also as substrate for lysoplasmalogenase, which liberates radioactive aldehydes at the same rate as from lysoplasmal ogen. Because the apparent Km and Vmax values are nearly identical for both substrates, the enzyme activities are inhibited in the same way, and the pH optimum is about 7.2 in both cases, it is concluded that both substrates were attacked by the same enzyme. The enzyme does not differentiate between a substrate substituted at the sn‐3 position of glycerol and one that is not. It requires only a free OH group at the sn‐2 position. Phosphoethanolamine phosphatase activity was also determined under our experimental conditions.

On the Phospholipid Metabolism of Glial Cell Primary Cultures..: II. Metabolism of 1-Alkyl-Glycerophosphoethanolamine During Time Course

Abstract: Primary cultures were prepared from newborn rat brain. After 16‐18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1‐[1‐3H]alkyl‐sn‐glycero‐3‐phosphoethanolamine (1‐alkyl‐GPE), for 1–20 h. Five main products were formed: 1‐alkyl‐2‐acyl‐GPE; 1‐alkyl‐2‐acyksn‐glycero‐3‐phosphocholine (1‐alkyl‐2‐acyl‐GPC); 1‐alkenyl‐2‐acyl‐GPE (ethanolamine plasmalogen); 1‐alkenyl‐2‐acyl‐GPC (choline plasmalogen); and 1‐alkyl‐glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl‐, 27.5% alkyl‐acyl‐, and 46.0% alkenyl‐acyl‐ compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1‐alkyl‐glycerol was a minor reaction.

Some Properties of Lysoplasmalogenase and Alkenylhydrolase from Rat Liver Microsomes

The existence of lysoplasmalogenase acting on ethanolaminelysoplasmalogen (1-alk-1’enyl-sn-glycero-3-phosphoethanolamine: alkenyl GPE) is now well established and is shown to occur in rat liver (Gunawan and Debuch, 1981) and rat brain (Gunawan and Debuch, 1982). We used a substrate 14C labeled within the aldehyde residue. Thus the radioactive aldehyde,liberated by the enzyme from the lyso compound was isolated as well as GPE in a 1:1 molar ratio (Gunawan and Debuch, 1985).