scholarly journals Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane-bound ribosomal particles.

1975 ◽  
Vol 67 (1) ◽  
pp. 16-24 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Messenger Rna ◽  
Specific Radioactivity ◽  
Kinetic Studies ◽  
Chain Termination ◽  
High Turnover ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
High Specific Radioactivity ◽  
Membrane Bound ◽  
Kinetics Of

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.

scholarly journals Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

1975 ◽  
Vol 67 (1) ◽  
pp. 25-37 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Membrane Fraction ◽  
Polypeptide Chain ◽  
Salt Treatment ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
Membrane Bound ◽  
Made In

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.

scholarly journals Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes.

1981 ◽  
Vol 88 (1) ◽  
pp. 42-50 ◽  
Author(s):  
B Mechler
Keyword(s):  
Polypeptide Chain ◽  
Mrna Translation ◽  
Normal Growth ◽  
Kinetic Studies ◽  
Myeloma Cells ◽  
Nascent Polypeptide ◽  
Membrane Attachment ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Mouse Myeloma

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N-terminal amino acid sequence of the nascent polypeptide chain.

Palmitate transport and fatty acid transporters in red and white muscles

1998 ◽  
Vol 275 (3) ◽  
pp. E471-E478 ◽  
Author(s):  
A. Bonen ◽  
J. J. F. P. Luiken ◽  
S. Liu ◽  
D. J. Dyck ◽  
B. Kiens ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Incubation Temperature ◽  
Kinetic Studies ◽  
Fatty Acid Transport ◽  
Fatty Acid Binding ◽  
Membrane Bound ◽  
Palmitate Uptake ◽  
Kinetics Of ◽  
Fold Excess ◽  
Sarcolemmal Vesicles

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPcconcentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (−50%), sulfo- N-succinimidyl oleate (−43%), anti-plasma membrane-bound FABP (FABPpm, −30%), trypsin (−45%), and when incubation temperature was lowered to 0°C (−70%). Palmitate uptake was also reduced by excess oleate (−65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was ∼6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates ( P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

scholarly journals Kinetic studies in the chemistry of rubber and related materials. I. The thermal oxidation of ethyl linoleate

1946 ◽  
Vol 186 (1005) ◽  
pp. 218-236 ◽  
Keyword(s):  
Thermal Decomposition ◽  
Reaction Mechanism ◽  
Thermal Oxidation ◽  
Molecular Oxygen ◽  
Kinetic Studies ◽  
Ethyl Linoleate ◽  
Chain Termination ◽  
Radical Chain ◽  
Kinetics Of ◽  
Sole Product

The kinetics of the initial stages of the thermal oxidation of ethyl linoleate (by molecular oxygen) have been investigated in the temperature range 35—75° C. From consideration of chemical and kinetic evidence the reaction mechanism has been established: oxidation chains are initiated by thermal decomposition of ethyl linoleate hydroperoxide (which in the early stages of oxidation is substantially the sole product). The chain propagation reactions are identified as R — + O 2 → R O 2 — and R O 2 — + R H → R OOH + R — (where R H represents ethyl linoleate). Chain termination occurs by mutual destruction of the radical chain carriers, R — and R O 2 — .

Kinetic Studies in the Chemistry of Rubber and Related Materials. II. The Kinetics of Oxidation of Unconjugated Olefins

1947 ◽  
Vol 20 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J. L. Holland ◽  
Geoffrey Gee
Keyword(s):  
Thermal Decomposition ◽  
Kinetic Studies ◽  
Chain Termination ◽  
Low Oxygen ◽  
Chain Reaction ◽  
Kinetics Of Oxidation ◽  
Chain Initiation ◽  
A Chain ◽  
Kinetics Of ◽  
Direct Attack

Abstract A brief review is given of kinetic work on the oxidation of representative mono, 1,4 and 1,5 olefins. The essential process in each case is identified as a chain reaction in which hydrocarbon radicals are formed, absorb oxygen, and then react with another molecule of olefin to give a hydroperoxide and a new free radical. Three methods of chain initiation are considered: (1) direct attack of oxygen on the olefin, (2) thermal decomposition of the hydroperoxide, (3) thermal decomposition of added benzoyl peroxide. Chain termination results from interaction of two free radicals; except at low oxygen pressures, these are both peroxidic.

scholarly journals Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

1976 ◽  
Vol 71 (1) ◽  
pp. 307-313 ◽  
Author(s):  
M Adesnik ◽  
M Lande ◽  
T Martin ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Fibroblasts ◽  
Ribosomal Subunits ◽  
Run Off ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
Extensive Release

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

scholarly journals The binding of ribosomal subunits to endoplasmic reticulum membranes

1972 ◽  
Vol 129 (3) ◽  
pp. 721-731 ◽  
Author(s):  
Francis S. Rolleston
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Messenger Rna ◽  
Mouse Liver ◽  
Peptide Bonds ◽  
Ribosomal Subunits ◽  
Membrane Bound

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60–80% of added 60S subunits and 10–15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane–60S subunit–40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.

scholarly journals The determination of lactate turnover in vivo with 3H- and 14C-labelled lactate. The significance of sites of tracer administration and sampling

1981 ◽  
Vol 194 (2) ◽  
pp. 513-524 ◽  
Author(s):  
J Katz ◽  
F Okajima ◽  
M Chenoweth ◽  
A Dunn
Keyword(s):  
Specific Radioactivity ◽  
Vena Cava ◽  
Turnover Rates ◽  
High Turnover ◽  
Glucose Carbon ◽  
Lactate Turnover ◽  
Carbon Recycling ◽  
Kinetics Of

L-[3-3H,U-14C]Lactate was administered to starved rats either as a bolus or by continuous infusion. Tracer administration was performed two ways: injection into the vena cava and sampling from the aorta (V-A mode), or injection into the aorta and sampling from the vena cava (A-VC mode). The specific-radioactivity curves after infusion or injection differed markedly with the two procedures. However, the specific radioactivities of 14C-labelled glucose derived from [U-14C]lactate were similar in the two modes. The apparent turnover rates of lactate calculated from the 3H specific-radioactivity curves in the V-A mode were about half those obtained from the 3H specific-radioactivity curves in the A-VC mode. The apparent contribution of lactate carbon to glucose carbon calculated from specific-radioactivity curves of the A-VC mode was greater than that obtained from the V-A mode. The apparent recycling of lactate carbon calculated from the specific radioactivities for [U-14C]- and [3-3H]-lactate was greater in the A-VC mode than the V-A mode. [U-14C] Glucose was administered in the two modes, but in contrast with lactate the specific radioactivities were only slightly different. An analysis to account for these observations is presented. It is shown that the two modes represent sampling from different pools of lactate. The significance of sites of tracer administration and sampling for the interpretation of tracer kinetics of compounds present in intracellular and extracellular spaces, and with a high turnover rate, is discussed. We propose that for such compounds, including lactate, alanine and glycerol, the widely used V-A mode leads to a marked underestimate of replacement, mass and carbon recycling, and that the A-VC mode is the preferred method for the assessment of these parameters.

scholarly journals Biogenesis of hepatocyte plasma-membrane domains. Incorporation of (3H)fucose into plasma-membrane and golgi-apparatus glycoproteins

1980 ◽  
Vol 192 (3) ◽  
pp. 903-910 ◽  
Author(s):  
W H Evans ◽  
N A Flint ◽  
P Vischer
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
Direct Transfer ◽  
Membrane Domains ◽  
Secretory Glycoproteins ◽  
Membrane Bound ◽  
Kinetics Of

1. Rats were injected intracaudally with [3H]fucose and its rate of incorporation into the fucoproteins of serum, Golgi and plasma-membrane subfractions was followed for up tp 2h. 2. Incorporation into the Golgi dictyosome and secretory-vesicular fractions reached a maximum at 15 min or less, but most of the radioactivity was associated with classes of secretory glycoproteins. Incorporation into sinusoidal plasma-membrane fractions reached a maximum at 30 min, coinciding with the maximum release of fucoproteins into the serum. Contiguous and canalicular plasma-membrane fractions were labelled slightly later and at a lower rate and specific radioactivity. 3. Fluorography of fucoproteins separated by polyacrylamide-gel electrophoresis helped to distinguish between the major secretory and membrane-bound glycoproteins. The results show that a major biogenetic sequence is probably from Golgi dictyosomes to Golgi secretory elements to a sinusoidal plasma membrane. 4. The kinetics of incorporation make it unlikely that there is rapid and direct insertion of glycoproteins into the bile-canalicular plasma membrane. A route involving direct transfer of glycoproteins via a membrane-mediated intracellular path from the blood sinusoidal to the bile-canalicular plasma membranes is proposed.

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