A Convenient Quick Method of Obtaining Vitamin B12 Concentrate

Borsook, Henry; Deasy, Clara L.; Haagen‐Smit, A. J.; Keighley, Geoffrey; Lowy, Peter H.

doi:10.1126/science.110.2864.528-a

Cited by 45 publications

(51 citation statements)

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“…In recent years, a number of studies have shown that the incorporation of labelled amino acids proceeds at a higher rate in microsome proteins than in the proteins of any other cell fraction both in ~vo (15)(16)(17)(18)(19)(20)22) and in vitro (21)(22)(23). This property was taken to suggest that the microsomes are active in protein synthesis and probably represent a cell organelle specialized in this direction.…”

Section: Introductionmentioning

confidence: 99%

Liver Microsomes

Palade

Siekevitz

1956

The Journal of Cell Biology

891

223

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Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO4-fixed, methacrylate-embedded tissue and pellets. It was found that most microsomes are morphologically identical with the rough surfaced elements of the endoplasmic reticula of hepatic cells. They appear as isolated, membrane-bound vesicles, tubules, and cisternae which contain an apparently homogeneous material of noticeable density, and bear small, dense particles (100 to 150 A) attached to their outer aspect. In solutions of various osmolar concentrations they behave like osmometers. The findings suggest that they derive from the endoplasmic reticulum by a generalized pinching-off process rather than by mechanical fragmentation. The microsome fractions contain in addition relatively few vesicles free of attached particles, probably derived from the smooth surfaced parts of the endoplasmic reticula. Dense, peribiliary bodies represent a minor component of the same fractions. The microsomes derived from 1 gm. wet weight liver pulp contained (averages of 10 experiments) 3.09 mg. protein N, 3.46 mg. RNA (RNA/protein N = 1.12), and 487 µg. phospholipide P. They displayed DPNH-cytochrome c reductase activity and contained an alcohol-soluble hemochromogen. The microsome preparations proved resistant to washing and "aging." Treatment with versene and incubation with ribonuclease (30 minutes at 37°C.) resulted in appreciable losses of RNA and in partial or total disappearance of attached particles. Treatment with deoxycholate (0.3 to 0.5 per cent, pH = 7.5) induced a partial clarification of the microsome suspensions which, upon centrifugation, yielded a small pellet of conglomerated small, dense particles (100 to 150 A) with only occasionally interspersed vesicles. The pellet contained ∼80 to 90 per cent of the RNA and ∼20 per cent of the protein N of the original microsomes. The supernatant accounted satisfactorily for the materials lost during deoxycholate treatment. The findings suggest that the microsomal RNA is associated with the small particles whereas most of the protein and nearly all of the phospholipide, hemochromogen, and DPNH-cytochrome c reductase activity are associated with the membrane or content of the microsomes.

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Section: Introductionmentioning

confidence: 99%

Liver Microsomes

Palade

Siekevitz

1956

The Journal of Cell Biology

891

223

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“…For this purpose the action of formic and performic acids (Peterson and Greenberg, 1952) and of sodium hydroxide Borsook, Deasy, Haagen-Smit, Keighley and Lowry, 1952) on the protein fractions from numerous experiments were examined. Table IV shows the percentage of specific radioactivity remaining after action of performic acid and of sodium hydroxide used under three different conditions.…”

Section: Mode Of Binding With Proteinsmentioning

confidence: 99%

The Distribution of Radioactivity in Tissues of the Rat Following the Administration of a Nitrogen Mustard Derivative

Cohn¹

1957

Br J Cancer

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AMINO ACIDS bearing the "nitrogen mustard" di-(2-chloroethyl)amino-side chain have been found to be effective inhibitors of the Walker carcinoma (Haddow, private communication; cf. Bergel and Stock, 1953). Their biological and chemotherapeutic behaviour is similar to that of other "nitrogen mustards" such-as di-(2-chloroethyl)methylamine itself. But special interest attached to one of these substituted amino acids, p-di-(2-chloroethyl)aminophenylalanine (Bergel and Stock, 1954), because its L-isomer was observed to be more active against the experimental tumour than the D-isomer (Haddow, private communication; cf. Bergel and Stock, 1953;Koller and Veronesi, 1956). This difference indicated that the configuration of the amino acid portion of the molecule might play a part in determining the mode of action of p-di-(2-chloroethyl)aminophenylalanine.The particular activity of the L-isomer, furthermore, suggested the possibility that this compound might influence protein formation or actually be incorporated into proteins. In the present investigation experiments were initially designed to test the occurrence of such incorporation with the aid of p-di-(2-chloroethyl)amino-DL-phenyl[fi-14C]alanine (PAM;Bergel, Burnop and Stock, 1955). In order to obtain precise information as to the origin of the different behaviours of the Dand L-forms it would clearly be desirable to do similar experiments with these forms. Unfortunately, the resolution of the DL-compound has not been achieved with the radioactively-labelled material. However, the work gave information in general on the distribution in the rat of a therapeutically effective nitrogen mustard derivative. MATERIALS AND METHODSAnimals and administration of PAM.-Male Wistar albino rats kept on a standard diet were injected intraperitoneally with PAM either suspended in arachis oil (8 mg./ml.) or as the sodium salt. This was freshly prepared by dissolving the acid in slightly less than the theoretical quantity of NaOH in methanol. The methanol was removed under slightly reduced pressure and NaCl

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“…Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide. Further operations were performed in the cold (1-4°).…”

Section: Methodsmentioning

confidence: 99%

Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

Freienstein¹,

Blobel²

1975

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

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The native small ribosomal subunit (S9) from rabbit reticulocytes which is able to initiate translation of globin mRNA in a cell-free system carries additional protein components. The latter can be separated from the subunit in a high salt sucrose gradient yielding a top-fraction (7) and a complex fraction (C), sedimenting at about 4 and 15 S, respectively. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed that fraction T contained four dominant polypeptides, while fraction C represents a large protein complex consisting of at least 10 polypeptides. SI' isolated from other sources showed similar patterns of their nonribosomal proteins.Reconstitution experiments revealed that fraction C is absolutely required for protein synthesis, while fraction T enhances protein synthesis only in the presence of C.The adherence of these protein factors to the subunit is not mediated by magnesium ions. Treatment of SO with EDTA and centrifugation in a magnesium-free sucrose gradient caused unfolding of the subunits and dissociation of several ribosomal proteins, but not of the factors. The unfolded ribosomal subunits sedimented as two distinct peaks. The more slowly sedimenting peak contained proteins of fraction T and the faster sedimenting one contained the 15S complex, indicating heterogeneity of the 9' population with respect to the factors attached to them.Initiation factors for protein synthesis in pro-and eukaryotes are in general found to be bound to ribosomes, from which they can be extracted by high concentrations of monovalent ions (1, 2). In eukaryotes it was recently established that the so-called native small ribosomal subunits (Sn), which comprise only a few percent of the total ribosomes, are able to initiate translation of mRNA in a reconstituted system containing derived large ribosomal subunits and pH 5 enzymes as the only other components (3, 4). Furthermore, it was reported that Sn contained several nonribosomal proteins (5) and that a salt extract from sn could stimulate globin mRNA translation in a cell-free protein synthesis system (6).These findings prompted us to identify the nonribosomal proteins found in Sn and to investigate their possible localization in other components of the ribosomal fraction, such as monosomes and polysomes, their mode of linkage to the small ribosomal subunit, their occurrence in a variety of cells, and their role in a reconstituted system. METHODSPreparation of Native Small Ribosomal Subunits (9'). Procedures were essentially as described (3). In brief: blood from anemic rabbits (7) was collected in the presence of cycloheximide. Further operations were performed in the cold (1-4°). Washed reticulocytes were lysed in 2 volumes of a solution of 10 mM Tris-HCl (pH 7.5)-10 mM KC1-1.5 mM MgCl2-2 mM dithiothreitol and homogenized with three strokes in a Potter-Elvehjem homogenizer. The postmitochondrial supernatant (10 min, 20,000 X gav) was centrifuged first for 30 min and the resulting supernatant was subsequently centrifuged again for 5 hr at 105,0...

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A Convenient Quick Method of Obtaining Vitamin B12 Concentrate

Cited by 45 publications

References 1 publication

Liver Microsomes

Liver Microsomes

The Distribution of Radioactivity in Tissues of the Rat Following the Administration of a Nitrogen Mustard Derivative

Nonribosomal proteins associated with eukaryotic native small ribosomal subunits.

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