scholarly journals DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

1966 ◽  
Vol 30 (3) ◽  
pp. 519-530 ◽  
Author(s):  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Pellet Fraction ◽  
Light Region ◽  
Centrifuge Tube ◽  
In Vivo Labeling

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

scholarly journals Mechanisms of hepatic phosphatidylcholine synthesis in the developing guinea pig: contributions of acyl remodelling and of N-methylation of phosphatidylethanolamine

1993 ◽  
Vol 290 (1) ◽  
pp. 67-73 ◽  
Author(s):  
G C Burdge ◽  
F J Kelly ◽  
A D Postle
Keyword(s):  
Fatty Acids ◽  
Guinea Pig ◽  
Molecular Species ◽  
Essential Fatty Acids ◽  
Specific Radioactivity ◽  
Long Chain Fatty Acids ◽  
Radioactivity Analysis ◽  
Species Specific ◽  
Phosphatidylcholine Synthesis

Hepatic phosphatidylcholine (PC) from the immature fetal guinea pig at day 55 of gestation comprised mainly unsaturated molecular species containing C18:2(n-6) and C22:6(n-3) at the sn-2 position, reflecting placental permeability to essential fatty acids. At both day 55 and term (day 68), [Me-14C]choline was incorporated in utero over 3 h largely into sn-1-C16:0 PC species, with incorporation into sn-1-C18:0 PC species increasing by 18 h of incubation. Comparison of specific radioactivities after 3 h and 18 h suggests PC acyl remodelling by phospholipase A1. No incorporation into C20:4(n-6)-containing PC species could be detected of either [Me-14C]choline in vivo or CDP-[Me-14C]choline in isolated microsomes. The major phosphatidylethanolamine (PE) species were 16:0/22:6 and 18:0/22:6. Although [14C]ethanolamine was initially incorporated mainly into sn-1-C16:0 species, specific-radioactivity analysis suggested differential turnover rather than acyl remodelling. [1,2-14C]Ethanolamine and [Me-14C]methionine incorporation into PC molecular species indicated that both newly synthesized and total PE pools were available for N-methylation. Since the PC pool synthesized from PE included C20:4- and C22:6-containing species, N-methylation may provide a mechanism for supplying essential long-chain fatty acids to developing tissues that can be regulated independently from bulk PC synthesis.

scholarly journals Therapy of cervical cancer using 131I-labeled nanoparticles

2018 ◽  
Vol 46 (6) ◽  
pp. 2359-2370 ◽  
Author(s):  
Wei Li ◽  
Danyang Sun ◽  
Ning Li ◽  
Yiming Shen ◽  
Yiming Hu ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Xenograft Model ◽  
Uv Spectrophotometry ◽  
Cytotoxicity Test ◽  
Mouse Xenograft Model ◽  
Late Apoptosis

Objective To evaluate the effectiveness of two kinds of Arg-Gly-Asp (RGD)-targeted 131I-containing nanoliposomes for the treatment of cervical cancer in vitro and in vivo. Methods The nanoparticle liposomes designated RGD-131I-tyrosine peptide chain (TPC)-L and 131I-RGD-L were prepared. The emulsion solvent evaporation method was used to encapsulate the polypeptide into liposomes. The quantity of entrapped polypeptide was measured using UV spectrophotometry. The labeling rates, radiochemical purities, and total radioactivities were measured using paper chromatography. Cytotoxicity was assessed using the MTS assay and flow cytometry. Therapeutic efficacy was monitored using a mouse xenograft model of cervical cancer. Results The labeling efficiency, radiochemical purity, and specific radioactivity of RGD-131I-TPC-L were greater than those of 131I-RGD-L. The cytotoxicity test indicated that late apoptosis of cells treated with RGD-131I-TPC-L and 131I-RGD-L was higher than that of cells treated with Na131I. The therapeutic effect of RGD-131I-TPC-L was better than that of 31I-RGD-L in the mouse model. Conclusions The specific activity of liposome-encapsulated RGD-131I-TPC-L was higher than that of 131I-RGD-L, which labeled liposomes directly. Moreover, the RGD-131I-TPC-L liposomes were more effective for killing xenografted tumor cells.

scholarly journals Activities of glucokinase and hexokinase in mammalian and avian livers

1984 ◽  
Vol 224 (2) ◽  
pp. 667-671 ◽  
Author(s):  
J C Stanley ◽  
G L Dohm ◽  
B S McManus ◽  
E A Newsholme
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Glycogen Synthesis ◽  
Hepatic Glycogen

A radiochemical assay for glucokinase activity was developed for use in high-speed supernatants of liver. The maximum activities of glucokinase ranged from 0.4 to 3.8 mumol/min per g fresh wt. at 30 degrees C in some avian and mammalian livers, including pigeon, guinea pig and man, in which previous reports indicated zero activities. The reported maximum rates of hepatic glycogen synthesis in livers of rat and man in vivo are similar to the calculated glucokinase activities at 10mM-glucose; therefore glucokinase activity should not limit glycogen synthesis from glucose.

scholarly journals Uptake of iodinated human kidney α-D-mannosidase by rat liver- Association with membrane elements and stability in vivo and in vitro

1976 ◽  
Vol 159 (3) ◽  
pp. 729-736 ◽  
Author(s):  
D V Marinković ◽  
S L Petrović ◽  
J V Martinović ◽  
J N Marinković
Keyword(s):  
Plasma Membranes ◽  
Specific Radioactivity ◽  
Human Kidney ◽  
Particulate Material ◽  
Enzymic Activity ◽  
Free Form ◽  
Rat Tissues ◽  
Liver And Kidney

1. Human kidney α-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.

Effects of fasting on flux and interconversion of leucine and alpha-ketoisocaproate in vivo

1981 ◽  
Vol 241 (1) ◽  
pp. E72-E75 ◽  
Author(s):  
S. Nissen ◽  
M. W. Haymond
Keyword(s):  
Specific Activity ◽  
Specific Radioactivity ◽  
Irreversible Loss ◽  
Transfer Rates ◽  
Leucine Metabolism

To determine directly the interconversion of circulating leucine and alpha-ketoisocaproate (KIC) in vivo, as well as the effects of fasting on leucine and KIC metabolism, 14- and 96-h fasted dogs were studied during simultaneous infusion of L-[4,5-3H]leucine and [U-14C]KIC. The specific radioactivities of 3H- and 14C-labeled leucine and KIC were determined and transfer rates calculated using a two-pool reversible model. In both groups, approximately 32% of the total leucine carbon entering was converted to KIC, whereas 60% of circulating KIC is converted to leucine. Plasma [3H]KIC specific radioactivity was only half of the circulating [3H]leucine specific activity, suggesting entry of KIC from an unlabeled pool. Fasting decreased the leucine-KIC interconversion, entry of unlabeled KIC, and irreversible loss of KIC. These data demonstrate that interconversion of circulating leucine and KIC is extensive and that fasting decreases the conversion of leucine to KIC in the labeled and unlabeled KIC pools, suggesting that transamination is a major regulator of leucine metabolism in fasting dogs.

scholarly journals Comparison of In Vitro and In Vivo Labeling of Virus-Induced L-Cell Interferon

1970 ◽  
Vol 56 (1) ◽  
pp. 134-148 ◽  
Author(s):  
D. Stanček ◽  
R. R. Golgher ◽  
K. Paucker
Keyword(s):  
Amino Acids ◽  
Electrophoretic Mobility ◽  
Specific Activity ◽  
Polyacrylamide Gels ◽  
Initial Activity ◽  
L Cell ◽  
In Vivo Labeling ◽  
Interferon Activity

Preparations of NDVuv-induced L-cell interferon were labeled in vitro with 125I and 3H gas, or in vivo through incorporation of amino acids-3H during synthesis. Prior to purification, more than 90% of the interferon titer was lost during in vitro labeling by either procedure, whereas 34% of the initial activity of in vivo-labeled material was preserved during preparatory handling. Purification by carboxymethyl-Sephadex chromatography and electrophoresis in polyacrylamide gels was about 100-fold, and electrophoretic profiles revealed close concordance between isotopes and interferon titers in all instances. Noninterferon proteins from control cells, although less extensively labeled with tritium during synthesis than proteins from interferon-producing cells and released in lesser amounts, also contained components of identical electrophoretic mobility and distribution in acrylamide gels as interferon. The highest specific activity (6 x 106 U/mg protein) but lowest cpm per interferon unit ratio (0.3) were exhibited by in vivo-labeled interferon. The advantage of better isotope incorporation through in vitro labeling techniques was largely offset by extensive losses in interferon activity.

scholarly journals The availability of carnitine acetyltransferase in mitochondria from guinea-pig liver and other tissues

1968 ◽  
Vol 110 (4) ◽  
pp. 739-746 ◽  
Author(s):  
P. J. Barker ◽  
N. J. Fincham ◽  
D C Hardwick
Keyword(s):  
Guinea Pig ◽  
Glutamate Dehydrogenase ◽  
Freeze Drying ◽  
Enzymic Activity ◽  
Carnitine Acetyltransferase ◽  
Cell Cytoplasm ◽  
Pig Liver ◽  
Triton X ◽  
Guinea Pig Liver

The carnitine acetyltransferase and glutamate dehydrogenase activities of guinea-pig liver and other tissues were estimated. Both enzymes are wholly mitochondrial, and can only be fully observed after disruption of the mitochondrion. Triton X-100 (0·1%) or freeze-drying revealed more activity than other methods tried. In mitochondria prepared and suspended in 0·25m-sucrose and in cell cytoplasm only small fractions of the total enzymic activity could be observed in guinea-pig liver: on average 7·5% of carnitine acetyltransferase and 5·5% of glutamate dehydrogenase. It is concluded that, in liver or mammary gland of goat, guinea pig or rat, little or no carnitine acetyltransferase is available in vivo to acetyl-CoA outside the mitochondrion.

scholarly journals Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1969 ◽  
Vol 113 (2) ◽  
pp. 387-397 ◽  
Author(s):  
M. J. Barnes ◽  
B. J. Constable ◽  
E. Kodicek
Keyword(s):  
Ascorbic Acid ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Specific Radioactivity ◽  
Cross Link ◽  
Control Groups ◽  
Acid Deficiency ◽  
Severe Impairment ◽  
Proline Incorporation

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.

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