Distributional changes of 32P-labeled acid-soluble phosphates and phospholipids among subcellular fractions during early vertebrate embryonic development

Early developing embryos of the toad Bufo arenarum Hensel were employed to study the content and in vivo labeling with ³²P of the acid-soluble phosphates and phospholipids at the subcellular level. The radionuclide was administered to the female toad along with the pituitary extract used to induce the ovulation.Most of the total phospholipids (68%) and proteins (84%) are confined to the yolk platelet fractions. Up to the heart beat stage (130 h of development) there are no significant changes detectable in protein and phospholipid content.The total P content in trichloroacetic acid-soluble fraction was distributed mainly between postmitochondrial supernatant (58%) and yolk platelet fraction (37%) in the unfertilized oocyte. As development proceeds an increase was observed in the former and a decrease in the latter. The acid-solube phosphates in the mitochondrial fraction only amount to 4% of the total embryo throughout the examined stages.The unfertilized oocyte contains about 98% of acid-soluble phosphates labeled with ³²P in the postmitochondrial supernatant and as development proceeds a striking decrease was found to occur while the radioactivity in the acid-soluble phosphates of mitochondrial and yolk platelet fractions increases significantly during the studied stages. About 11.5% of the lost radioactivity from the acid-soluble phosphates was found to be used to label the phospholipids.     

Distributional changes of P-labeled acid-soluble phosphates and phospholipids among subcellular fractions during early vertebrate embryonic development

Early developing embryos of the toad Bufo arenarum Hensel were employed to study the content and in vivo labeling with ³²P of the acid-soluble phosphates and phospholipids at the subcellular level. The radionuclide was administered to the female toad along with the pituitary extract used to induce the ovulation.Most of the total phospholipids (68%) and proteins (84%) are confined to the yolk platelet fractions. Up to the heart beat stage (130 h of development) there are no significant changes detectable in protein and phospholipid content.The total P content in trichloroacetic acid-soluble fraction was distributed mainly between postmitochondrial supernatant (58%) and yolk platelet fraction (37%) in the unfertilized oocyte. As development proceeds an increase was observed in the former and a decrease in the latter. The acid-solube phosphates in the mitochondrial fraction only amount to 4% of the total embryo throughout the examined stages.The unfertilized oocyte contains about 98% of acid-soluble phosphates labeled with ³²P in the postmitochondrial supernatant and as development proceeds a striking decrease was found to occur while the radioactivity in the acid-soluble phosphates of mitochondrial and yolk platelet fractions increases significantly during the studied stages. About 11.5% of the lost radioactivity from the acid-soluble phosphates was found to be used to label the phospholipids.     

Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.     

Phosphatidic acid, phosphatidylinositol, phosphatidylserine and cardiolipin in the course of early embryonic development. Fatty acid composition and content in whole toad embryos and in mitochondrial fractions

The fatty acid composition and content of phosphatidylinositol, phosphatidylserine and phosphatidic acid have been studied during the early development of toad embryos. Acidic phospholipids have been analyzed in whole oocytes and embryos and in the following subcellular fractions: yolk platelets, mitochondria and microsomes. Also cardiolipin, a mitochondrial phospholipid, has been analyzed. Gastrula stage embryos have shown, mainly in the mitochondrial fraction, an increase in the content of phosphatidic acid, phosphatidylserine and phosphatidylinositol with respect to unfertilized oocytes. Changes in the distribution of acyl groups of phosphatidic acid have been detected when different subcellular fractions are compared. On the other hand, the phosphatidylserine composition remains unmodified. Arachidonate and stearate are the principal components of phosphatidylinositol. Cardiolipin shows the same composition up to gastrulation and linoleate comprises about 50% of the total acyl groups.     

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.     

In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis

Abstract: Brains of 3-week-old C57BL/6J mice were homogenized and fractionated into several subcellular components, each of which was examined for ability to synthesize the myelin basic proteins (MBPs) in vitro. Myelin basic proteins were purified from incubation mixtures by conventional means. That the products of synthesis were the myelin basic proteins was established by solubility at pH 3, co-chromatography with authentic proteins on carboxymethylcellulose and co-migration with standards in two different polyacrylamide gel electrophoretic systems. The fractions examined for their ability to synthesize MBPs were the whole homogenate, postnuclear supernatant, postmitochondrial supernatant, crude mitochondrial pellet, free ribosomes and bound ribosomes. Although there was no requirement for exogenous energy sources for protein synthesis in the whole homogenate, as the homogenate was fractionated an increasing requirement emerged. Most of the label in the MBP preparations from whole homogenate and postnuclear supernatant incubations migrated with the large (L) and small (S) MBPs on gel electrophoresis; however, as the homogenate was subfractionated and incubated, a greater percentage of the label migrated more slowly than L and S on acetic acid-urea gels. To show synthesis of the MBPs the L and S bands were cut out of these gels and rerun on sodium dodecylsulfate gels. Alternatively, MBP preparations were subjected directly to two-dimensional gel electrophoresis and the bands corresponding to L and S were excised and counted. With this method only the whole homogenate, postnuclear supernatant, postmitochondrial supernatant and free ribosomes were observed to synthesize the MBPs in vitro. The "bound" ribosomes were not observed to synthesize significant amounts of the MBPs, incubated either intact or released from the membrane. It was concluded that the free ribosomes are the principal site of synthesis of the myelin basic proteins in the brain.     

Subcellular distribution of "peripheral type" binding sites for [3H]Ro5-4864 in guinea pig lung. Localization to the mitochondrial inner membrane

Binding of [3H]Ro5-4864, a specific ligand for "peripheral type" benzodiazepine receptors, was determined in subcellular fractions of guinea pig lung. Even though the level of binding was predominant in the mitochondrial fraction, nuclear and cytosolic fractions also contained significantly measurable amounts of binding sites. The presence of binding sites in the microsomal fraction and in a fraction intermediate in density between the mitochondria and microsomes depended on which buffer was used to homogenize the tissue. If calcium-containing mannitol buffer was used, binding was negligible in the postmitochondrial organelles. However, in the case of sucrose buffer which did not contain any calcium, the postmitochondrial organelle fractions contained measurable amounts of binding sites. Most probably, these binding sites were of mitochondrial and nuclear origin. Furthermore, binding sites in the mitochondria were associated with the succinic dehydrogenase-enriched mitochondrial inner membrane, but not with the monoamine oxidase- and cholinephosphotransferase-enriched outer mitochondrial membrane. Furthermore, several proteolytic enzymes caused a decrease in binding of the ligand to the mitochondrial membrane only under hypotonic conditions and not under isotonic conditions, suggesting that the location of the receptors is inside the mitochondria.     

ENZYME ACTIVITIES IN CHICK EMBRYONIC CARTILAGE : Their Subcellular Distribution in Isolated Chondrocytes

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.     

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.     

Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma.

1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.     

Purification and properties of alpha-aminoadipate aminotransferase from rat liver and kidney mitochondria.

Recently we reported an affinity chromatography method to purify alpha-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

The effect of adrenocorticotrophin on protein degradation in rat adrenal gland and liver

The rate of adrenal protein degradation appears to be slower in rats to which ACTH (adrenocorticotrophin) has been chronically administered. As measured by the exponential decay of radioactively labelled adrenal protein in vivo, the mean half-lives of total protein and of mitochondrial, microsomal and 18000g-supernatant protein were significantly longer in ACTH-treated animals. Experiments in which either [(3)H]leucine or NaH(14)CO(3) was used to label proteins showed that of the fractions studied, the effect on mitochondrial protein degradation was most pronounced. The half-lives of the same subcellular fractions in rat liver were not affected by ACTH. The possibility that the results might have been caused by changes in radioisotope reutilization and pool size is discussed.     

Change in phosphatase and acid cathepsin activity in the dog cerebral cortex in terminal states]

Acid phosphatase, alkaline phosphatase and acid cathepsin activity increased in the subcellular fraction of the grey matter of dog brain in proportion to the duration of compression ischemia, particularly in the postmitochondrial supernatant. An increase in the alkaline phosphatase and of the acid cathepsin activity in the postmitochondrial supernatant was also observed at the period of clinical death caused by blood loss and preceded by hypotension of different duration and various levels of arterial pressure. Comparison of the above data indicated a common mechanism of proteolysis activation in the nervous tissue in the terminal states under study.     

Lipid composition of subcellular particles of human blood platelets

Human platelets can be fractionated into three main subcellular components: granules, membranes, and a soluble fraction. In this study we determined the phospholipid and neutral lipid content of the granules and membranes. Quantitative relationships between lipids and protein were examined. The fatty acid and aldehyde composition of individual phospholipids and neutral lipids was also determined. Whole platelets had a lower lipid to protein ratio than did the subcellular particles, but the basic lipid composition of the granules, membranes, and platelets was similar. The phospholipid composition of platelets and subcellular fractions was found to differ only in that granules had a lower percentage of lecithin. Each of the phospholipid classes displayed a distinctive fatty acid pattern which was the same in all fractions and in whole platelets. The major neutral lipid was free cholesterol. Cholesteryl esters, triglycerides, and free fatty acids were minor components. The molar ratio of cholesterol to phospholipid in the platelet membranes was lower than that of brain myelin and erythrocyte ghosts. Some differences in fatty acid composition of the neutral lipids of platelet fractions were found. A special lipid composition or constituent that would correlate with platelet function has not been found.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Manganese, copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle

To clarify trace element distribution in red and white muscle, and to verify two populations of muscle mitochondria, the iron, zinc, copper, and manganese concentrations of whole muscle and their subcellular fractions were determined. The iron, zinc, copper, and manganese concentrations of red muscle were 1.83, 4.31, 2.05, and 1.67 times higher than those of white muscle, respectively. In skeletal muscle subcellular distribution or iron, zinc, and copper were entirely different and that of manganese was relatively similar as compared with those in liver reported previously. The pattern of mineral distribution in all fractions of red muscle was similar to that of white muscle, but their concentrations in some fractions were different between red and white muscle, e.g., iron, zinc, and manganese in supernatant fraction and copper in nuclear and microsomal fractions. The difference between subsarcolemmal and interfibrillar mitochondria were ascertained by the distribution of trace elements.     

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.     

The lipid composition of ovine placental tissue homogenate and its subcellular fractions

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.     

Purification and Properties of α-Aminoadipate Aminotransferase from Rat Liver and Kidney Mitochondria

Abstract

Recently we reported an affinity chromatography method to purify α-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.