Carotenoids in fish II. Carotenoids and vitamin A in some fishes from the coastal region of the Black Sea

The presence of various carotenoids and vitamin A in seven species of fish from the coastal region of the Black Sea was investigated by means of columnar and thinlayer chromatography. The investigations revealed the presence of the following carotenoids: Mugil auratus: ß-carotene, canthaxanthin, lutein, zeaxanthin, astaxanthin ester and astacene. Diplodus annularis: ß-carotene, canthaxanthin, tunaxanthin, lutein, zeaxanthin and astacene. Diplodus sargus: ß-carotene, tunaxanthin, lutein, taraxanthin, zeaxanthin and astaxanthin. Crenilabrus tinca: tunaxanthin, canthaxanthin, lutein, astaxanthin and astacene. Blennius sphinx: ß-carotene, χ-carotene (?), lutein, tunaxanthin, taraxanthin and astaxanthin. Blennius sanguinolentus: ß-carotene, tunaxanthin and astaxanthin (ester and free). Gobius melanostomus: ß-carotene and astacene. Some fractions were not identified. Vitamin A was found in all species investigated.     

Uptake of [3H]vitamin D3 from low and high density lipoproteins by cultured human fibroblasts

The plasma distribution and cellular uptake of [3H]vitamin D3 was studied in vitro using cultured human fibroblasts. Incubation of [3H]vitamin D3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [3H]1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [3H]vitamin D3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D3 was time, concentration, and temperature dependent. At a concentration of 50 micrograms LDL/ml of medium, the uptake of [3H]vitamin D3 from LDL at 37 degrees C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.     

The effect of vitamin E on the intracellular distribution of the different oxidation states of selenium in rat liver

1. The liver intracellular distribution of (75)Se, (75)Se(2-) and (75)SeO(3) (2-) formed from orally administered Na(2) (75)SeO(3) was studied in rats given four different dietary treatments. 2. Subcellular fractionation was done by using sucrose density gradients in a B XIV zonal centrifuge rotor, and conditions were established so that separation of lysosomal, mitochondrial, smooth- and rough-surfaced endoplasmic reticulum, and soluble fractions was achieved. 3. Marker enzymes acid phosphatase, succinate-2 - p - iodophenyl - 3 - p -nitrophenyl - 5 - phenyltetrazolium reductase and glucose 6-phosphatase were used, together with electron microscopy, to establish the identity of the fractions. 4. The dietary treatments investigated were: (a) vitamin E-deficient diet for 3 months, re-fed with vitamin E during the terminal 5 days; (b) vitamin E-deficient diet; (c) adequate diet; (d) vitamin E- and selenium-deficient diet, re-fed with vitamin E during the terminal 5 days. 5. In adequately fed rats, selenide was particularly associated with the mitochondrial fractions; in vitamin E-deficient rats, little selenide was found and the buoyant density of the mitochondria was increased, whereas re-feeding with vitamin E showed a restoration of the normal pattern. In vitamin E- and selenium-deficient rats, re-fed with vitamin E, there was no tendency for selenide to be localized in the mitochondria. 6. In the microsomal regions of the gradients, adequately fed rats showed a concentration of selenide, particularly in the smooth endoplasmic reticulum fractions, and to a lesser extent in the rough endoplasmic reticulum fractions. This was not observed in vitamin E-deficient rats, and the normal pattern was restored on re-feeding with vitamin E, both in rats given the vitamin E-deficient diet and the vitamin E- and selenium-deficient diet. 7. Some selenide was also found in the soluble fractions, when vitamin E was present, and a substantial proportion of this selenide was found to pass through a dialysis membrane. 8. These results are taken to support our hypothesis that the active form of selenium may be selenide located in non-haem iron-containing proteins, and that the function of vitamin E may be to protect the selenide from oxidation.     

Carotenoids and Vitamin A in the Crabs Pachygrapsus marmoratus (FABRE) and Eriphia spinifrons (HERBST)

Investigations have been carried regarding carotenoids and vitamin A in the crabs Pachygrapsus marmoratus (FABRE) and Eriphia spinifrons (HERBST) from the Black and Adriatic Sea. The presence of carotenoids and vitamin A was determined by means of column and thinlayer chromatography. The following carotenoids were found: Pachygrapsus marmoratus: β-carotene, cryptoxanthin, canthaxanthin, lutein, astaxanthin and vitamin A; Eriphia spinifrons: β-carotene, echinenone, canthaxanthin, isozeaxanthin, zeaxanthin, lutein (ester and epoxy), astaxanthin and astacene.     

Distribution of dipeptidase activity between lysosomes and soluble fraction of rat liver.

Dipeptidase activity toward Arg-Phe, Arg-Gly, and Trp-Leu exhibited bimodal distribution in the lysosomal and soluble fractions of rat liver. The majority (50-70 percent) of the dipeptidase activity was present in the soluble fraction. Some evidence for a plasma membrane dipeptidase, which hydrolyzes Trp-Leu but not Arg-Phe or Arg-Gly, also was found. The lysosomal dipeptidase activity had a pH optimum of 6.0-7.0, and was activated by sulfhydryl reagents. Lysosomal localization for some of the dipeptidase activity was established with Triton WR-1339 fractionation and latency experiments.     

Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.     

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Fatty acid, carotenoid and vitamin A composition of tissues of free living gulls

The aim of this study was to investigate fatty acid and carotenoid profile as well as vitamin A (retinol and retinol esters) content in gull (Larus fucus) tissues. Palmitic (16:0) and stearic (18:0) fatty acids were major saturates in all the tissues studied. Oleic acid (18:1n-9) was the major monounsaturate in the tissue phospholipids varying from 11.9% (liver) up to 18.2% (lung). Arachidonic acid (20:4n-6) was the major unsaturate in the phospholipid fraction in all the tissues. Liver contained the highest total carotenoid concentration which was 5 and 7 fold higher compared to kidney and pancreas. In the liver beta-carotene was major carotenoid. In contrast, in all other tissues beta-carotene was minor fraction with lutein being major carotenoid. Zeaxanthin, canthaxanthin, beta-cryptoxanthin and echinenone were also identified in the gull tissues. Liver and kidney were characterised by the highest vitamin A concentrations (1067.5 and 867.5 microg/g, respectively). Retinol comprised from 55.3% (pancreas) down to 8% (kidney) of the total vitamin A but was not detected in the abdominal fat. Retinyl palmitate was the major retinyl ester in the liver, kidney and heart (44.2; 38.1 and 46.0% of total retinyl esters). In muscles and abdominal fat retinyl stearate was the major retinyl ester fraction. Therefore high proportions of beta-carotene were found in gull liver and peripheral tissues were enriched by lutein and zeaxanthin compared to the liver, a very high concentration of retinyl esters in the kidney was observed and tissue-specificity in retinyl ester proportions in peripheral tissues was found.     

A one-step technique for the subcellular fractionation of total cell homogenates

A procedure was developed for the rapid, analytical subcellular fractionation of entire homogenates from the Chinese hamster ovary and HeLa cell lines. The procedure avoids a nuclear sedimentation step and the losses that accompany such a step. A key to the development of this procedure was the addition to homogenates of either micrococcal nuclease or DNase I. Nuclease-treated homogenates were fractionated on self-forming Percoll gradients. The entire procedure from cell harvesting through collecting gradient fractions took only 2.5 h. The position of marker enzymes in the gradient fractions indicated clear resolution of plasma membranes, Golgi apparatus, endoplasmic reticulum, and lysosomes. This procedure should facilitate many studies requiring subcellular fractionation of cultured cells.     

Subcellular localization of adenylate cyclase in thymocytes

In almost all cell types, adenylate cyclase is located in the plasma membrane. In lymphocytes, however, this enzyme has been claimed to be largely present in intracellular compartments. In this study, the distribution of adenylate cyclase activity in subcellular fractions of calf thymocytes was reinvestigated by a balance sheet approach. When subcellular fractionation was performed in the absence of ATP and dithiothreitol, less than a half of the homogenate basal activity could be recovered in the fractions, and this amount was distributed almost equally in three main compartments: the plasma membrane fraction, the microsomal and mitochondrial fractions and the nuclear fraction. However, if enzyme activity in the above fractions was measured in the presence of the stimulatory agents NaF, guanylylimidophosphate or guanosine 5'-O-(3-thio)triphosphate, or if the subcellular fractionation was performed in media containing ATP and dithiothreitol, the overall recovered activity greatly increased (up to 90%) and the distribution was shifted in favour of the plasma membrane fraction (up to 65% of the recovered activity). The adenylate cyclase properties were similar in all fractions. The ionophore alamethicin did not alter the subcellular distribution of the enzyme. The localization of adenylate cyclase in thymocytes thus appears to be primarily, if not uniquely, in the plasma membrane, as generally found in other cell types.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [³H]retinol and ¹⁴C-labelled vitamin D-3, ¹⁴C-labeled vitamin D-3 appeared later in the intestinal lymph than [³H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Vitamin K-dependent carboxylase: Evidence for cofractionation of carboxylase and epoxidase activities,and for carboxylation of a high-molecular-weight microsomal protein

The vitamin K-dependent carboxylase from rat liver microsomes has been fractionated by submitting a crude preparation of this activity to chromatography on different column supports. A constant ratio of vitamin K epoxidation and vitamin K-dependent carboxylation was observed in all column fractions with good carboxylase activity, supporting the hypothesis that these two activities are carried out by the same enzyme complex. The preparation obtained (Complex B) is stable for several days when left on ice and has the same general properties as those observed in Triton X-100-solubilized microsomes. When antiserum raised against Complex B was incubated with Complex B, a twofold increase in carboxylase activity was observed. Benzidine staining showed that an appreciable pool of the antibody population was directed against hemeprotein(s). These data and spectral analyses indicated that a major contaminant of the preparation in cytochrome P-450. Although endogenous prothrombin precursors were absent in the crude starting preparation, a constant ratio of endogenous substrate carboxylation and carboxylation of a soluble substrate was observed during fractionation. A protein with a molecular weight of approximately 120,000 which copurified with Complex B was identified as substrate for the carboxylase.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine. Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [3H]retinol and 14C-labelled vitamin D-3, 14C-labeled vitamin D-3 appeared later in the intestinal lymph than [3H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.     

The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells.

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.     

Cellular fractionation and isolation of the plasma membrane of Burkitt's lymphoma cells

A procedure for cellular fractionation and preparation of plasma membrane from a Burkitt's lymphoma cell line is described. This procedure involves homogenization with a Polytron in buffered isotonic sucrose, and separation of cellular fractions by differential and isopycnic centrifugation in sucrose. The isolated plasma membrane fraction contains 44% of the cellular cholesterol, 50% of the ouabain-sensitive (Na⁺ + K⁺)-ATPase activity, 43% of the γ-glutamyltranspeptidase activities and 16% of the phospholipid. This fraction contains only 3% of cellular protein and is contaminated with less than 4% of the total cellular activities of microsomal, lysosomal, mitochondrial, Golgi and soluble marker enzymes. The cholesterol : phospholipid molar ratio of the crude plasma membrane is 0.56. The membranes in this fraction are in the form of vesicles. Further purification of plasma membrane is achieved by sucrose density gradient centrifugation and results in a 25- to 30-fold enrichment of plasma membrane markers. Plasma membrane markers band in these gradients between 1.10 and 1.15 g/cm³.The distribution patterns in the cell fractions of 18 cellular constituents are quantitatively determined. Most constituents are found to distribute in a fashion consistent with the results obtained in other systems. Thymidine-5′-phosphodiesterase (phosphodiesterase I), esterase, nucleoside diphosphatase and glucose-6-phosphatase, however, are shown to be poor markers of membrane fractions in this system.Lactoperoxidase-catalyzed iodination was used to identify several plasma membrane proteins which are exposed at the surface. After separation of labeled polypeptides by sodium dodecyl sulfate gel electrophoresis, the predominant labeled protein was identified as the heavy chain of IgM. Several lesser labeled proteins were observed.     

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.     

A comparison of colicines K and V extracted from solid medium

Following the extraction of colicine K and colicine V from digest nutrient agar, the crude colicine was divided into 3 portions. Each portion was subjected to a primary treatment with either 30% chloroform, 90% acetone or 66% absolute alcohol. Aliquots of the active fractions obtained from each of these primary treatments were subsequently exposed to either of the other two extracants. It was noted that the best method for primary purification of both colicines was blending with chloroform but the results of subsequent fractionation differed. Colicine K was insoluble in 66% absolute alcohol whereas colicine V remained soluble in alcohol but was precipitated by 90% acetone.Mouse toxicity tests revealed that the toxic fraction of the crude colicine V was precipitated by 66% alcohol and that the non-toxic fraction, soluble in alcohol, was associated with the activity of the colicine. All the active fractions of crude colicine K were lethal for mice.We wish to express our appreciation to the various colleagues who were kind enough to send the colicinogenic or the indicator strains used in this investigation. This research was assisted in part by a grant HERT 215 from the Scottish Hospital Endowment Research Trust and partly by a grant from the Duff Fund. We are grateful to Mrs. P. Hercus for technical assistance.     

Fractionation and characterization of normal rabbit plasma proteins

1. An adaptation of the low-temperature low-salt ethanol procedure for the fractionation of rabbit plasma proteins into six fractions is described. 2. The composition of the fractions and the distribution of haptoglobins, caeruloplasmin and transferrin were determined. The protein and protein-bound carbohydrate distribution in the fractions is similar to that of human plasma proteins separated by a similar procedure. 3. The purification of albumin, α₁-acid glycoprotein, transferrin and γ-globulin was carried out.