scholarly journals Neuronal N-acetyl-β-D-glucosaminidase. Evidence for its biosynthesis in vitro

1976 ◽  
Vol 158 (3) ◽  
pp. 513-527 ◽  
Author(s):  
J A Khawaja ◽  
O Z Sellinger
Keyword(s):  
Concanavalin A ◽  
Microsomal Fraction ◽  
Deae Cellulose ◽  
Neuronal Cell ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Freezing And Thawing ◽  
Cellulose Acetate Electrophoresis ◽  
Fluorescent Band

Neuronal cell bodies, isolated in bulk from 8-day-old rat cerebral cortices, were incubated in the presence of a 3H-labelled amino acid mixture, and subcellular fractions isolated by differential centrifugation. The particulate fractions were frozen/thawed in 0.20 M-sucrose/0.1 M-KCl [Selling et al. (1973) Biochim. Biophys. Acta 315, 128-146] and the profiles of acid-insoluble radioactivity and N-acetyl-beta-D-glucosaminidase (glucosaminidase) activity compared in the resulting non-sedimentable fractions by DEAE-cellulose chromatography and cellulose acetate electrophoresis. Radioactivity and glucosaminidase activity co-migrated to a significant extent. Electrophoresis revealed that after 1 min of incubation 42% of the radioactivity of the non-sedimentable microsomal fraction after freezing and thawing co-migrated with an intensely fluorescent band of glucosaminidase activity. Since the pellet fraction obtained on freezing/thawing the microsomal fraction contained up to 75% of the RNA, 95% of the radioactivity and 45% of the glucosaminidase, a detailed study of the association between its radioactivity and nascent glucosaminidase activity was undertaken. After 1 and 2 min of incubation, followed by centrifugation of the microsomal pellet on 35-60% (w/v) sucrose density gradients, radioactivity and glucosaminidase activity exhibited parallel profiles in the region of heavy polyribosomes and at the top of the gradient which contains spontaneously released nascent polypeptide chains. DEAE-cellulose chromatography of these chains revealed glucosaminidase A to be the principal nascent glucosaminidase component, with glucosaminidases B and C as minor peaks. After 2 min of incubation, all of the glucosaminidase components appeared labelled, and glucosaminidase A exhibited two distinct sub-components. The pattern of glucosaminidase labelling in the soluble and microsomal fractions suggested that newly formed glucosaminidase molecules traverse both the cellular sap and the lumen of the endoplasmic reticulum. Only glucosaminidase A reacted specifically with concanavalin A and radioactive glucosaminidase A could be successfully regenerated by treatment with alpha-methyl-D-glucoside. Glucosaminidase A and a substantial portion of the radioactivity associating with it could be readily converted into glucosaminidase B by re-chromatography on DEAE-cellulose and by reaction of the concanavalin A-glucosaminidase A complex with methyl glucosides.

FACTORS INFLUENCING THE NUTRITIONAL VALUE OF FISH FLOUR: III. FURTHER STUDIES ON AVAILABILITY OF AMINO ACIDS

1963 ◽  
Vol 41 (7) ◽  
pp. 1589-1594 ◽  
Author(s):  
A. B. Morrison
Keyword(s):  
Amino Acids ◽  
Nutritional Value ◽  
Chloride Content ◽  
In Vitro Digestion ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Deficient Diet ◽  
Weight Gains ◽  
Flour Sample

Weight gains of male weanling rats given fish flour sample X were significantly increased by addition to the diet of methionine, histidine, threonine, and tryptophan. When histidine or methionine were omitted from the amino acid mixture, weight gains were similar to those found with the unsupplemented flour, and the combination of methionine and histidine was as effective as the four amino acids. Supplements of histidine and methionine had no effect on weight gains of rats given fish flour sample CFF, which was of high nutritional value. Sample X contained methionine in an amount similar to that of sample CFF, and somewhat less histidine. The amounts of methionine and histidine released during in vitro digestion with pancreatin were much less for sample X than for sample CFF. Steaming sample X for 30 minutes significantly increased its gross protein value determined in a methionine-deficient diet, but had no effect on the total or organic chloride content. It was concluded that sample X contained unavailable methionine and histidine.

The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

scholarly journals Dietary amino acids: effect of depletion and recovery on synthesis in vitro in rat skeletal muscle and liver

1974 ◽  
Vol 31 (1) ◽  
pp. 67-76 ◽  
Author(s):  
P. T. Omstedt ◽  
Alexandra Von Der Decken
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Energy Levels ◽  
Wheat Gluten ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Amino Acid Mixtures ◽  
Wet Weight ◽  
Ribosomal Activity

1. Rats were given diets containing 200 g/kg of a complete or incomplete amino acid mixture or of high- or low-quality proteins. After 6 d the amino acid-incorporating activity of ribosomes from skeletal muscle and liver was studied.2. The level of isotope incorporation relative to ribosomal RNA was similar for casein supplemented with methionine and for a complete amino acid mixture with the composition of whole-egg protein. Per wet weight of tissue there was a significant decrease after feeding with the complete amino acid mixture.3. There was a significant decrease in activity after feeding with amino acid mixtures deficient in lysine, methionine or tryptophan. In skeletal muscle, but not in liver, the ribosomal activity was less than that obtained with wheat gluten. Activity per wet weight of both tissues was less than that obtained with wheat gluten.4. Refeeding with methionine for 1 d resulted in complete restoration of ribosomal activity and activity per wet weight in skeletal muscle.5. After lysine deficiency, protein synthesis per unit wet weight of both tissues and ribosomal activity in liver were not restored after 2 d of refeeding. Recovery of ribosomal activity in skeletal muscle was complete after 1 d.6. Rats receiving the 200 g casein/kg diet supplemented with methionine at daily energy levels of 263, 176, 141 and 106 KJ (62.6, 42.1, 33.7 and 25.3 kcal) showed no changes in ribosomal activity, but there was a significant decrease in activity per wet weight when 106 KJ were given.

Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum

1983 ◽  
Vol 61 (12) ◽  
pp. 1304-1314 ◽  
Author(s):  
David R. Allred ◽  
Irwin W. Sherman
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Acetylcholinesterase Activity ◽  
Amino Acid Mixture ◽  
Malarial Parasite ◽  
Acid Mixture ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Developmental Modulation

Under conditions of in vitro culture, Plasmodium falciparum incorporated amino acids into particulate (membrane) and soluble proteins in a pattern which changed sequentially and which was dependent upon the stage of parasite maturation. Synchronized cultures pulse labeled with a mixture of 15 14C-labeled amino acids or [14C]histidine alone displayed stage-related patterns of polypeptide biosynthesis. Certain plasmodial proteins were associated with both particulate (membrane) and soluble fractions, whereas others appeared to be specific to a given fraction. Proteolysis of intact infected cells with pronase under conditions which removed 97 ± 2.2% of the endogenous red cell acetylcholinesterase activity did not cause the apparent removal of any radiolabeled proteins; this suggests the absence of externally exposed, parasite-synthesized proteins in the infected red cell membrane. Such a result was consistent whether the radiolabel was [14C]histidine or the 14C-labeled amino acid mixture. These results indicate that specific modulation of parasite biosynthetic patterns occurs during the asexual reproductive cycle and is probably one mechanism whereby parasite differentiation occurs. Despite the formation of surface excrescences on infected red cells containing mature parasites, results of surface digestion experiments failed to demonstrate the presence of surface-exposed plasmodial proteins.

Evidence for Active Transport of the Dipeptide Carnosine (β-Alanyl-l-Histidine) by Hamster Jejunum in Vitro

1974 ◽  
Vol 46 (6) ◽  
pp. 693-705 ◽  
Author(s):  
D. M. Matthews ◽  
Jill M. Addison ◽  
D. Burston
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Transport ◽  
Amino Acid Mixture ◽  
Intestinal Wall ◽  
Free Form ◽  
Acid Mixture ◽  
Total Uptake ◽  
Hydrolysis Of

1. The characteristics of intestinal transport and hydrolysis of carnosine (β-alanyl-l-histidine) have been studied in rings of everted hamster jejunum in vitro. 2. During incubation with carnosine, large amounts of intact peptide appeared in the intestinal wall, accompanied by small amounts of the constituent amino acids in the free form. Although there was some extracellular hydrolysis, the free amino acids appearing in the intestinal wall were almost entirely derived from intracellular hydrolysis of the peptide. Incubation in l-alanyl-l-histidine resulted in uptake of the constituent amino acids in the free form without appearance of intact peptide in the intestinal wall. 3. Total uptake of β-alanine (both peptide-bound and free) and total uptake of histidine were greater from a low concentration (1 μmol/ml) of carnosine than uptake of these amino acids from the equivalent amino acid mixture. At a high concentration of carnosine (20 μmol/ml), total uptake of β-alanine was greater from the peptide than from the equivalent amino acid mixture but total uptake of histidine was less. At this concentration, total uptake of β-alanine plus total uptake of histidine from the peptide was approximately the same as from the amino acid mixture. 4. Uptake of carnosine by jejunal rings was the result of a saturable process (Kt 9·4 μmol/ml, Vmax. 2·7 μmol g−1 initial wet wt. min−1). Intact carnosine was concentrated in the intestinal wall, the concentration ratio between intracellular fluid and incubation medium being up to 3·4/1. Uptake of carnosine was reduced by anoxia, metabolic inhibitors and replacement of medium Na+. Na+-dependent active transport was shown to be involved in uptake of carnosine by hamster jejunum in vitro.

MAINTENANCE OF ASCARIS LUMBRICOIDES IN VITRO: II. CHANGES IN MUSCLE AND OVARY CARBOHYDRATES

1963 ◽  
Vol 41 (1) ◽  
pp. 1673-1689 ◽  
Author(s):  
R. P. Harpur
Keyword(s):  
Amino Acid ◽  
Ascaris Lumbricoides ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Mean Values ◽  
The Third ◽  
Intestinal Nematode ◽  
Fresh Material ◽  
Vitamin Mixture

The possibility of using carbohydrate determinations to assess the nutritional state of the intestinal nematode, Ascaris lumbricoides, is discussed and investigated. For fresh material the following mean values and standard deviations were found: muscle glycogen (I) 14.8 ± 2.5 g%, muscle trehalose (II) 1.11 ± 0.35 g%, ovary glycogen (III) 7.2 ± 0.6 g%, and ovary trehalose (IV) 0.27 ± 0.13 g%. A single replication of a 3 × 25 design, with confounding, was used to study the effects of 1, 2, and 3 days in vitro together with N2 versus 5% O2 and the presence and absence in the keeping medium of 14 mM NH4+, 0.2% glucose, a vitamin mixture, and an amino acid mixture. None of these substances affected the drop to 11.0 g% which occurred in I on the first day and only glucose showed a highly significant sparing action which was manifest on the second and third day. Glucose also had a significant sparing action with III but mainly on the third day. Highly significant decreases occurred in both II and IV on the second day.

scholarly journals PROTEIN SYNTHESIS IN SYNAPTOSOMAL FRACTIONS

1972 ◽  
Vol 52 (3) ◽  
pp. 526-535 ◽  
Author(s):  
P. Gambetti ◽  
L. A. Autilio-Gambetti ◽  
N. K. Gonatas ◽  
B. Shafer
Keyword(s):  
Protein Synthesis ◽  
Specific Activity ◽  
Mitochondrial Protein ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Mitochondrial Protein Synthesis ◽  
Synaptosomal Fraction ◽  
Tritiated Amino Acids ◽  
Vitro Protein

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30–50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75–77% of the total processes. 34–45% of the grains were related to processes containing ribosomes which accounted for only 4–7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.

scholarly journals The cell-free synthesis of cytochrome c by a microsomal fraction from rat liver

1971 ◽  
Vol 124 (4) ◽  
pp. 685-694 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Juan P. Ortega ◽  
Magally González
Keyword(s):  
Cytochrome C ◽  
Microsomal Fraction ◽  
Freezing And Thawing ◽  
Prosthetic Group ◽  
Extraction And Purification ◽  
Isolated Liver ◽  
Cytoplasmic Ribosomes ◽  
Homogenization Medium

Conditions were investigated for demonstrating the synthesis in vitro of the complete molecule of cytochrome c by isolated liver microsomal systems from partially hepatectomized rats. It was first found that in vivo the early labelled cytochrome c associated with the microsomal fraction required, by comparison with the mitochondrial pool, more drastic conditions of extraction and its binding was less affected by freezing and thawing of the subcellular particles. The procedure of extraction and purification of cytochrome c had to be modified accordingly, to assure the recovery of the recently synthesized molecule. Several subcellular fractions were isolated from regenerating liver with a homogenization medium containing either 5 or 10mm-Mg2+ and most of them were active in the synthesis of the cytochrome c apoprotein. The microsomal fraction, in the presence of either cell sap or pH5.0 fraction, was also able to incorporate [59Fe]haemin, δ-amino[3H]laevulic acid and 55Fe into the prosthetic group of cytochrome c. These experiments confirm firmly the conclusions of our previous results obtained in vivo showing that both the apoprotein and the haem moieties are made and linked together on cytoplasmic ribosomes and only then is the complete molecule transferred to the mitochondria.

A Serum-Free in vitro Culture System for Crayfish Organs

1986 ◽  
Vol 41 (4) ◽  
pp. 472-476 ◽  
Author(s):  
Gerd Gellissen ◽  
Marco Traub ◽  
Klaus-Dieter Spindler
Keyword(s):  
Culture System ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Serum Free Medium ◽  
Free Medium ◽  
Astacus Leptodactylus ◽  
Serum Free ◽  
In Vitro Culture System

Midgut gland and hypodermis of the crayfish Astacus leptodactylus have been cultured in a serum-free medium for several days. The medium consists of 1 part of van Harreveld solution and 1 part of an amino acid mixture supplemented with 1.2 mᴍ Na2HP04, 12 mᴍ Hepes and 80 mᴍ glucose. The antibiotics penicillin (15 mg/l) and streptomycin (25 mg/1) were added for long term culturing. This medium, called TG medium, allows the maintainance of the tissues for more than 100 h without any loss of their viability with respect to protein synthesis and secretion.

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