Analysis of C14-labeled proteins by disc electrophoresis

Fairbanks, Grant; Levinthal, Cyrus; Reeder, Ronald H.

doi:10.1016/0006-291x(65)90589-9

Cited by 459 publications

(95 citation statements)

References 3 publications

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“…Proteins 11 and 17 appear to be internal vesicle proteins by our criteria. From the data presented here it is difficult to know if these proteins are peripheral to, are integral parts of, or cross the membranes.…”

Section: Resultsmentioning

confidence: 82%

See 1 more Smart Citation

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

Wagner¹,

Kelly²

1979

Proc. Natl. Acad. Sci. U.S.A.

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Intact synaptic vesicles prepared from the electric organ of the marine elasmobranch Narcine brasifiensis have eight major polypeptides demonstrable on sodium dodecyl sulfate gels. Six of these copurify with the synaptic vesicles during isolation of vesicles Ey chromatography on CPG-3000 and, by this criterion, are specific to vesicles. The other two are either shared by many membranes or are contaminants. One of these proteins comigrates with actin. Three different approaches were used to determine which proteins were exposed on the external, cytoplasmic surface of the vesicle and which were internal. The first was susceptibility to the proteases trypsin, Streptomyces griseus protease, and Pronase; the second was labeling by e membrane-impermeable reagent diazotized [15I~iodosulfanilic acid; and the third was iodination catalyzed by lactoperoxidase. In general, the three approaches give the same result: six of the eight proteins are on the external, cytoplasmic surface and two are accessible only after the vesicles are lysed by freezing and thawing or by detergents. Five of the vesicle-specific proteins are external and one is internal. The actin-like protein is internal. Proteins involved in the interaction of vesicles with the presynaptic membrane during exocytosis might be expected to be vesicle specific and external.

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Section: Resultsmentioning

confidence: 82%

“…Lysis does not occur during the experimental manipulations since less than 5% of the contents are lost. The strongest argument, however, for the absence of lysed vesicles is the large increase in the availability of proteins 11 and 17 to membrane-impermeable reagents after artificial lysis.…”

Section: Resultsmentioning

confidence: 99%

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

Wagner¹,

Kelly²

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…The material was analysed on 10% acrylamide gels [8] containing 0.05% SDS after mixing with dansylated SFV structural proteins. The positions of the radioactive bands were determined by autoradiography after drying down longitudinal gel slices [9].…”

Section: Cell-free Protein Synthesismentioning

confidence: 99%

In vitro synthesis of structural proteins of semliki forest virus directed by isolated 26 S RNA from infected cells

Clegg

Kennedy

1974

FEBS Letters

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“…After 1 h of dedifferentiation, the mitochondria incorporated sion in 1.5 mi of 2 % sodium dodecylsulfate and then sequentially sonicated for 2 min, heated to 95 "C for 10 min, incubated at 37 "C for 2 h, centrifuged, and dialysed. The proteins were electrophoresed on 10 % acrylamide gels containing 0.1 % sodium dodecylsulfate, and autoradiographs of the gels were prepared by the methods of Fairbanks et al [18]. (A) This figure shows strips of autoradiographs of polypeptides from cells to which ~-[U-'~C]leucine (316 mCi/ mmol) was added to a final concentration of 11.9 pM after the 15 min preincubation.…”

Section: Resultsmentioning

confidence: 99%

“…Electrophoresis was stopped when the bromophenol blue tracking dye had migrated about 7 cm ( E 9 h), and the gels were fixed by placing them overnight in 10 % trichloroacetic acid containing 10 mM L-leucine at 0 "C. The gels were then prepared for autoradiography by the procedures of Fairbanks et al [18]. Exposures of the dried gels, sliced longitudinally, were made on X-ray film held tightly in place with a press.…”

Section: Incorporation Of (14c]~eucine By Mitochondria In Vivomentioning

confidence: 99%

Biogenesis of Mitochondrial Membranes in Neurospora crassa

Hawley

Greenawalt

1975

European Journal of Biochemistry

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The conidia of Neurospora crassa entered logarithmic growth after a 1‐h lag period at 30°C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3‐h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide‐sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0–8‐h‐old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 μg cycloheximide/ml, increased from low levels in the conidia to peak levels at 3–4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0–4‐h‐old cells, but declined after 15 min of incorporation in 6 and 8‐h‐old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12200 to 80000). Within the high‐molecular‐weight class, proteins of between 12000 and 21500 molecular weight were preferentially labelled early in germination, whereas after 8 h of growth proteins of 27500 to 80000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000‐molecular‐weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.

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Analysis of C14-labeled proteins by disc electrophoresis

Cited by 459 publications

References 3 publications

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle.

In vitro synthesis of structural proteins of semliki forest virus directed by isolated 26 S RNA from infected cells

Biogenesis of Mitochondrial Membranes in Neurospora crassa

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