Becca Fleischer

Some studies on the metabolism of phospholipids in Golgi complex from bovine and rat liver in comparison to other subcellular fractions

Abstract 1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria, and nuclei from bovine liver were isolated and the purity assessed using specific marker enzymes. 2. 2. Cholinephosphotransferase and acyl-CoA:1,2-diacyl-sn-glycerol acyltransferases were found to be absent in the Golgi complex, mitochondria, plasma membrane, and nuclei. The de novo synthesis of lecithin and triacylglycerols is apparently localized exclusively in the smooth and rough endoplasmic reticulum. 3. 3. Acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase was found to be localized mainly in rough and smooth microsomes from bovine and rat liver; though also significant activity was found in the Golgi complex of bovine liver. On the other hand, no significant acylation of 1-acyl-sn-glycero-3-phosphorylcholine could be shown in the Golgi system from rat liver. 4. 4. Phosphatidylglycerol synthesis was found to take place predominantly in rat liver mitochondria. Significant synthesis of this phospholipid was, however, also found in rough and smooth microsomes and in Golgi of rat liver. 5. 5. The Golgi complex of rat liver was shown to contain both phospholipase A1 and A2 activities acting on exogenous phosphatidylethanolamine, the activity of the former being somewhat larger.

Lipid composition of the golgi apparatus of rat kidney and liver in comparison with other subcellular organelles

Golgi apparatus isolated from both rat liver and rat kidney have been characterized with respect to their neutral and phospholipid content and their phosphopipid composition and compared with mitochondria, rough endoplasmic reticulum and plasma membranes. In addition, the distribution of sulfatide in the subcellular fractions of rat kidney was determinich are rich in cholesterol esters and ubiquinone. Removal of about 75% of the cisternal contents of rat liver Golgi reduced its content of cholesterol esters but not of ubiquinone. The Golgi complex of liver most closely resembles endoplasmic reticulum in its phospholipid composition except for a higher content of sphingomyelin. Removal of most of the contents of the Golgi cisternae did not appreciably alter the phospholipid composition of the Golgi apparatus of liver. Goligi apparatus from kidney has a phospholipid composition which resembles liver Golgi much more closely than it does any other cell fraction from kidney. The sulfatide content of kidney Golgi, the cell fraction richest in this glycolipid, is about 14% of the total lipid present in this fraction. Sulfatide was present in plasma membranes, mitochondria and rough microsomes, but at about one-third the level found in Golgi. Sulfatide is the main glycosphingolipid present in all the cell fractions from kidney which were studied.

Biosynthesis of lipids in golgi complex and other subcellular fractions from rat liver

Abstract 1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria and nuclei from rat liver were isolated and their purity assessed using specific marker enzymes. 2. 2. The various subcellular fractions were assayed for the following processes: biosynthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine, the conversion of phosphatidylserine into phosphatidylethanolamine, the formation of lecithin via N-methylation, and the activation of palmitic and octanoic acids. 3. 3. None of these processes were found to be present in Golgi complex. 4. 4. The endoplasmic reticulum appears to be the principal site in the cell for the synthesis of sphingomyelin, CDPdiglycerides, phosphatidylinositol, phosphatidylserine and the formation of lecithin. Interestingly, the biosynthesis of phosphatidylserine appears to be four times more active in rough than in smooth microsomes, which might suggest a ribosomal localization of this process. 5. 5. Except for CDPdiglyceride synthesis, mitochondria do not contain any of the synthesizing activities described in 4. Mitochondria are, however, the only site in the cell where phosphatidylserine is decarboxylated. This activity appears to be localized in the inner membrane. 6. 6. The activation of palmitate is localized predominantly in endoplasmic reticulum and mitochondria, though some activity was detected in plasma membranes as well. All other cell organelles, including Golgi and probably nuclei, did not contain significant palmitoyl-CoA synthetase activity. The subcellular distribution of octanoyl-CoA synthetase resembled that of palmitoyl-CoA synthetase except that the former enzyme is more active in mitochondria than in microsomes.

Preparation and characterization of golgi membranes from rat liver

Abstract 1. 1. A Golgi-rich fraction from rat liver has been isolated by the direct application of the zonal centrifugation method used previously to isolate Golgi-rich membranes from bovine liver. 2. 2. UDP-galactose -N- acetylglucosamine galactosyl transferase is concentrated about 100-fold in this fraction compared to the homogenate and appears to be a useful marker enzyme for this organelle in rat liver as well as beef liver. As in bovine liver Golgi, the fraction from rat liver is only slightly contaminated by other organelles as evidenced by low glucose-6-phosphatase, ATPase and acid phosphatase activities. Thiamine pyrophosphatase activity was found present in endoplasmic reticulum and plasma membranes as well as Golgi membranes from rat liver, and was therefore not a useful marker. 3. 3. Some species differences in the Golgi preparations exist. Rat liver Golgi contain little or no rotenone-insensitive NADH- or NADPH-cytochrome c reductase activity whereas bovine liver Golgi have significant levels of these activities. In addition, the Golgi fraction from rat liver appears to have a unique and characteristic protein profile after electrophoresis in polyacrylamide gels as compared with endoplasmic reticulum, plasma membranes and mitochondria. Bovine liver Golgi preparations, on the other hand, appear very similar to endoplasmic reticulum after electrophoresis on acrylamide gels. 4. 4. A major protein of band mobility 0.456 ± 0.008 , relative to ribonuclease A, is present in rat liver Golgi preparations. This band is also characteristic of bovine liver Golgi preparations and may be due either to serum very low density lipoproteins or serum albumin, or both. 5. 5. Using the information obtained from this type of preparation, a simpler, one-step procedure was devised which increases the yield of Golgi membranes from rat liver to 0.15–0.3 mg Golgi protein per g as compared to 0.05 mg Golgi protein per g obtained by the zonal procedure.

Glycosidase activity of bovine liver plasma membranes

1. 1. Plasma membranes were isolated on a large scale from bovine liver by a modified method of Neville1. They were characterized with respect to contamination by endoplasmic reticulum, lysosomes, and mitochondria. 2. 2. The membranes were found to contain glycosidases capable of hydrolyzing uridine diphosphogalactose (UDP-Gal) and uridine diphosphoglucose as well as o-nitrophenyl α- or β-galactosides. 3. 3. The hydrolysis of UDP-Gal using a 14C-labeled substrate in the galactose moiety allowed a rapid estimation of the amount of plasma membrane contaminating microsomal fractions. Smooth microsomes were contaminated about 14% with plasma membranes. This value agreed with estimates based on the content of Mg2+-stimulated ATPase and of 5′-nucleotidase. Rough microsomes contained about 5% plasma membranes using this criterion. Purified nuclei and mitochondria from bovine liver showed negligible amounts of this activity. 4. 4. The optimum pH of the enzyme was 7.2 using cacodylate buffer. Mg2+ stimulated the activity slightly whereas Mn2+ was inhibitory. The Km for UDP-Gal was 0.33 mM.

Localization of cerebroside — Sulfotransferase activity in the Golgi apparatus of rat kidney

Abstract A Golgi-rich fraction that contains both uridine diphosphogalactose: N-acetylglucosamine galactosyltransferase activity and 3′-phosphoadenosine-5′-phosphosulfate:cerebroside sulfotransferase activity has been isolated from rat kidney. Both activities are increased about 80-fold in the Golgi fraction compared to the homogenate. Little or no galactosyltransferase or sulfotransferase activity was found in purified nuclei, mitochondria, rough endoplasmic reticulum, plasma membranes and supernatant. The results indicate that both galactosyltransferase and sulfotransferase are localized in Golgi apparatus from rat kidney. This is the first evidence that Golgi apparatus functions to modify a lipid component of the cell.

Cytochrome b5 and P-450 in liver cell fractions.

The contiguous membranes of liver, i.e. nuclei, rough and smooth microsomes, plasma membranes, and Golgi vesicles, were analyzed for cytochromes and P-450 content. Only cytochrome b5 and P-450 were detectable. The highest cytochrome b5 and P-450 content was in the microsome fraction. The Golgi vesicle contains cytochrome b5 but there is little or no P-450. Thus P-450 levels can be used to discriminate between microsomes and Golgi comples. The plasma membrane is practically devoid of cytochromes.

The distribution of albumin precursor protein and albumin in liver

Abstract Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.

Gel electrophoresis patterns of the proteins of organelles isolated from bovine liver.

Abstract Mitochondria, smooth and rough microsomes, Golgi vesicles, and plasma membranes have been isolated from bovine liver and their protein components separated by polyacrylamide gel electrophoresis. Characteristic patterns were obtained for each organelle studied. The mobilities of the major protein bands relative to ribonuclease A were determined and comparisons made among the organelles. The major protein components of rough and smooth microsomes were indistinguishable. Golgi vesicles were similar to, but easily distinguishable from, endoplasmic reticulum (as represented by the rough microsome fraction). A major band in the Golgi preparation had the same mobility as the major protein component found in serum β-lipoproteins ( d ). All the organelles except plasma membranes had a major protein component with mobility identical to that found for purified “structural protein” isolated from mitochondria.

The nucleotide content of rat liver Golgi vesicles

Abstract The nucleotide profile of rat liver Golgi vesicles isolated using sucrose gradients has been determined by high-pressure liquid chromatography. The nucleotide composition of this Golgi preparation, probably modified by osmotic shock, differs from that of liver supernatant fraction and from isolated rough microsomes. The major nucleotides present in the Golgi have been tentatively identified as uridine diphosphate and a peak containing uridine monophosphate plus cytidine monophosphate at 1.6 and 0.5 nmol/mg protein, respectively. In order to minimize osmotic shock, we have modified the isolation of Golgi using D 2 O-sucrose gradients. Intact Golgi from these gradients were extracted directly and analyzed. Higher levels of nucleotides were found in the unshocked preparation, and the profile was also altered, although it was still distinct from that of liver supernatant. Four major peaks were found, tentatively identified as uridine monophosphate plus cytidine monophosphate, adenosine monophosphate, UDP, and uridine diphosphogalactose plus uridine diphosphoglucose, at 6.4, 6.4, 6.1, and 3.3 nmol/mg protein. These results indicate that the membrane of the Golgi apparatus is not freely permeable to nucleotides but that selective mechanisms exist for the uptake or exclusion of specific nucleotides from this organelle. The fact that UDP is selectively retained in shocked Golgi vesicles may indicate the presence of a binding protein which would prevent interference of Golgi function by UDP, a highly inhibitory product of galactosyltransferase.