Subcellular and generic distribution, molecular weights, and proportions of oligopeptides

Swank, Richard T.; Sheir, Geraldine I.; Munkres, Kenneth D.

doi:10.1021/bi00797a021

Cited by 36 publications

(33 citation statements)

References 28 publications

(12 reference statements)

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“…Electrophoresis was performed at 25 mA/slab for 4-5 h at 20°C. Actin (42 000 daltons), beef cardiac myosin light chain 1 (22 000 daltons) and the cyanogen bromide peptides of myoglobin (see [6] for molecular masses) were run simultaneously in the second dimension as a guide to the approximate molecular masses of the peptides. The slab gels were stained with Coomassie brilliant blue by the Weber and Osborn method [7].…”

Section: Limo Dimensional Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Atrial and ventricular cardiac myosins contain different heavy chain species

Flink

Banerjee

et al. 1978

FEBS Letters

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Section: Limo Dimensional Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Atrial and ventricular cardiac myosins contain different heavy chain species

Flink

Banerjee

et al. 1978

FEBS Letters

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“…Three sets of fractions were pooled; the first two were re-chromatographed to yield pure subunit I and 11 + 111: the third fraction was used as the starting material for the guanidine-HC1 isolation of subunits V, V1 and VII. Subunits I1 and TIT were separated by dodecyl sulphate gel electrophoresis in the presence of urea [16]. The twice chromatographed fraction containing pure subunits I1 + I11 was pooled, concentrated and electrophoresed on 12 cylindrical gels.…”

Section: Subunits Of Cytochrome C Oxidasementioning

confidence: 99%

“…Subunits I1 and 111 not only co-migrate on dodecyl sulphate gcl clcctrophoresis (see below) but also appeared as a single subunit with identical molecular weight in gcl filtration chromatography in the presence of dodecyl sulphatc [I 51. Only when electrophoresed in the presence of urea [16] did subunit I11 migrate differently from subunit I1 [25]. The fraction that contained subunits IV to VII (routinely, the fractions were examined by gel electrophoresis for identification of the subunits) was used for isolation of individual subunits V. VI and VTT [15,25].…”

Section: Isolution Of Cytochrome Oxiduse Subunitsmentioning

confidence: 99%

Immunological Studies on Cytochrome c Oxidase: Arrangements of Protein Subunits in the Solubilized and Membrane‐Bound Enzyme

Chan

Tracy

1978

European Journal of Biochemistry

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Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, I1 and 111) and guanidine hydrochloride (subunits V, VT and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly crosslinked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = 1, 36700; 11, 24300; 111, 20400; IV, 17300; V, 12300; VI, 8700; and VII, 5100. Monospecific rabbit antisera were obtained against subunits 1, IV, V, VI and V11 and a mixture of subunits I1 and 111. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits I1 + 111, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25 % inhibition while anti-I immunoglobulin did not inhibit the activity.The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(I1 + 111) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using '251-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits I1 and 111 are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and V1 are buried within the membrane, not exposed on either side.Cytochrome c oxidase, the terminal enzyme of the electron transport chain, is an integral component of the mitochondrial inner membrane. Earlier studies on the structure of the enzyme indicated that it isa complex unit containing two hemes a, two copper atoms and multiple dissimilar protein subunits [I -31. Experiments using antibodies against the native oxidase [4,5] and chemical labelling with bovine heart mitochondria [4,6] have shown that cytochrome oxidase spans the mitochondrial membrane asymmetrically, which is the basis of the chemiosmotic hypothesis .of oxidative phosphorylation. Based on differential reactivities of normal and inverted mitochondrial membranes towards surface-labelling reagent, Eytan et al. ourselves that the interpretation based on chemical labelling experiments does not suffer from relatively low specific activity of labelled products as well as possible damaging effects on membranes by the chemical labels, we decided to use individual subunitspecific antibodies to establish the arrangements of cytochrome c oxidase subunits in mitochondrial membrane. It is generally acknowledged that antibody molecules ...

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“…Polyacrylamide gel electrophoresis was carried out either in a continuous pH system on 15-cm-long gels containing 12.5 % acrylamide, 0.6 % N,N'-methylenebisacrylamide, 8 M urea and 0.1 % sodium dodecylsulfate [13] or, for a better separation of the structuraI proteins, in a discontinuous pH system on 10-cmlong gels containing 10 % acrylamide, 0.25 % N,N'-methylenebisacrylamide and 0.1 % sodium dodecyl-…”

Section: Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

A Suppressor of Nonsense Mutations in Bacillus subtilis

Camacho¹,

Carrascosa²,

Viñuela³

et al. 1974

European Journal of Biochemistry

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The suppressor-sensitive mutants of phage 429, susB47 and susN212, do not induce the synthesis of proteins NP1 and 11, respectively, when they infect Bacillus subtilis su-. A new polypeptide of molecular weight 75000, smaller than that of protein NP1, is produced in susB47-infected cells. An analysis of the methionine-containing tryptic peptides of protein NP1 and the 75000-molecularweight polypeptide showed that both proteins are related. When mutants susB47 and susN212 infect B. subtilis su+3, a small amount of proteins NP1 and 11, respectively, is synthesized, the level of the suppression being about 10 %. The results indicate that the B. subtilis su+3 strain is a suppressor of nonsense mutations.Nonsense mutants are very useful to detect protein gene products as under restrictive conditions a complete protein is not synthesized; instead, a fragment of length related to the position of the mutation is produced [I]. In Escherichia coli several strains which suppress the nonsense codons are available [2-41.More recently several suppressor strains from Bacillus subtilis have been isolated [5 -71 but there is no direct evidence as to the nature of the suppressor. By using asporogenous derivatives of the B. subtilis strain Mu8u5u5 su+3 isolated by Georgopoulos [6], two collections of suppressor-sensitive mutants of bacteriophage 429 have been isolated [8,9]. In this paper we show that a sus mutant of 429 produces, in B. subtilis su-, a fragment of the normal protein. This mutation, as well as others, are partially suppressed when the mutants are grown in the suf3 strain of B. subtilis, giving rise to the appearance of a small amount of the normal phage proteins. These results indicate that the B. subtilis su+3 strain [6] contains a suppressor of nonsense mutations. MATERIALS AND METHODS Bacteria and PhageThe non-permissive host B. subtilis 168 try-spoOAllONA [lo] [lo], also obtained from Dr P. Schaeffer.The suppressor-sensitive (sus) mutants B47 and N212 were from the collection of Moreno et al. [9]. The preparation of phage stocks and the phage assays were as described by Talavera et al. [ll]. Ultraviolet Irradiation and Labelling of BacteriaThe bacteria were grown in minimal medium and irradiated with ultraviolet light for 6 or 7.5 min prior to infection as described by Carrascosa et al. [12]. The irradiated bacteria were resuspended, at 5 x 108 cells/ ml, in minimal medium containing 0.5 mM amino acids except leucine which was 0.01 mM. Aliquots of the irradiated bacteria were infected with wildtype phage or with the mutants susB47 or susN212 at a multiplicity of 20 and shaken at 37 "C. The bacteria infected with the sus mutants were labelled with [3H]leucine (25 pCi/ml; 0.023 mM) from 20 to 23 min after infection; at 23 min a 100-fold excess of nonradioactive leucine was added and the incubation was continued for 2 min. Wild-type infected and uninfected bacteria were pulse-labelled at the same times with [14C]-leucine (7.5 pCi/ml; 0.023 mM) and chased with nonradioactive leucine for 2 min. To follow phage de...

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Subcellular and generic distribution, molecular weights, and proportions of oligopeptides

Cited by 36 publications

References 28 publications

Atrial and ventricular cardiac myosins contain different heavy chain species

Atrial and ventricular cardiac myosins contain different heavy chain species

Immunological Studies on Cytochrome c Oxidase: Arrangements of Protein Subunits in the Solubilized and Membrane‐Bound Enzyme

A Suppressor of Nonsense Mutations in Bacillus subtilis

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