ABSTRACT
Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.
Subject(s)
Hepatocytes/metabolism , Peroxisomes/metabolism , Phosphatidylinositols/biosynthesis , 1-Phosphatidylinositol 4-Kinase/metabolism , Animals , Cells, Cultured , Endoplasmic Reticulum/metabolism , In Vitro Techniques , Phosphatidylinositol Phosphates/biosynthesis , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/metabolism , Rats , Subcellular Fractions/metabolismABSTRACT
Low-temperature adaptation and cryoprotection were studied in the lactic acid bacterium Lactococcus lactis MG1363. An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 degrees C for 4 h compared to mid-exponential-phase cells grown at 30 degrees C, indicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L. lactis MG1363 (Wouters et al., 1998, Microbiology 144, 2885-2893). The 7 kDa CSPs appeared to be the most strongly induced proteins upon cold shock to 10 degrees C. Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 degrees C, while induction was lower at 20 and 4 degrees C. However, pre-incubation at 20 and 4 degrees C, as well as stationary-phase conditions, also induced cryoprotection (approx. 30-, 130- and 20-fold, respectively, compared to 30 degrees C mid-exponential phase). For all treatments leading to an increased freeze survival (exposure to 4, 10 and 20 degrees C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested. Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions. However, the level of observed freeze protection does not directly correlate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied. This revealed that L. lactis cells overproducing CspD at 30 degrees C show a 2-10-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that other factors supply additional cryoprotection.
