Starch-gel electrophoresis—Application to the classification of pituitary proteins and polypeptides

Ferguson, K. A.

doi:10.1016/s0026-0495(64)80018-4

Cited by 980 publications

(425 citation statements)

References 9 publications

Supporting

Mentioning

409

Contrasting

Unclassified

Order By: Relevance

“…For three protein standards and for all three polypeptides of citramalate lyase, linear Ferguson plots [33] intersecting at the same point (Ox acrylamide) were obtained (Fig. 5).…”

Section: Molecular Properties Of Citrumulute Lyasementioning

confidence: 82%

The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

Buckel

Bobi

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

1. The enzyme citramalate lyase from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCI, and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsirlla aerogenes.2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using iod0[2-'~C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism.3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. uerogmes. The molecular weight of citramalate lyase is equal to that of citrate lyase ( M , = 5.2-5.8 x lo5) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: CI 5 . 3 -5 . 6~1 0~; fi 3 . 3 -3 . 6~1 0~; 7 1 . 0 -1 . 2~ lo4) in probably equal molar amounts. These data lead to a hexameric structure (~( , f l , y )~ of the complete enzyme.4. Pantothenate (5 mol/mol of enzyme) and the essential cystearnine residues were exclusively present in the y-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the a and 8-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier.5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.

show abstract

“…For three protein standards and for all three polypeptides of citramalate lyase, linear Ferguson plots [33] intersecting at the same point (Ox acrylamide) were obtained (Fig. 5).…”

Section: Molecular Properties Of Citrumulute Lyasementioning

confidence: 82%

The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

Buckel

Bobi

1976

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The migration of the purified enzyme was analysed on a series of denaturing and native gels containing different polyacrylamide concentrations and the apparent size of the enzyme was determined by the method of Ferguson (1964). Under denaturing conditions the estimated molecular mass of CA is about 27 kDa while under native conditions the molecular mass is approximately 54 kDa, suggesting that the native enzyme is a dimer.…”

Section: Properties Of the Purified Enzymementioning

confidence: 99%

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

et al. 2000

View full text Add to dashboard Cite

Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified S2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to α-type CAs. The upstream region of acaP was fused to the lacZ gene and β-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC 50 in the range of 10 N8 M, and in the culture media concentrations as low as 30 µM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP ::Kan r mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO 2 gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO 2 and that there is no expression under aerobic conditions.

show abstract

“…by comparison to the mobility of cytochrome c. Ferguson plots of log10 R F against gel concentration for each protein gave straight lines which intersected at the same point when extrapolated to zero gel concentration, indicating that all the proteins, including the TGMV capsid polypeptide, migrated in a standard manner (Ferguson, 1964). The gradient of the Ferguson plots, called the retardation coefficient (KR), is a function of the protein size (Rodbard & Chrambach, 1970).…”

mentioning

confidence: 98%

Identification of the Coat Protein Gene of Tomato Golden Mosaic Virus

Kallender¹,

Petty²,

Stein³

et al. 1988

Journal of General Virology

View full text Add to dashboard Cite

SUMMARYAn open reading frame, designated AR1, on DNA component A of tomato golden mosaic virus has been identified as the virus coat protein gene on the basis of the molecular weight and amino acid composition of the virus capsid polypeptide, the mass spectra and N-terminal sequences of peptides produced by cyanogen bromide cleavage of the capsid polypeptide and by the binding of antibodies, induced by immunization of a rabbit with a fl-galactosidase-AR1 fusion protein, to the capsid polypeptide in a Western blot.Tomato golden mosaic virus (TGMV) belongs to the Geminivirus group of plant viruses, members of which are characterized by double-icosahedral particles and genomes of circular ssDNA (Hamilton et al., 1981 ;Matthews, 1982). Nucleotide sequence analysis of the infectious cloned DNA components of TGMV, DNA A and DNA B, has identified six open reading frames (ORFs) with the potential to code for proteins with 100 amino acids or more (Hamilton et al., 1984). Four of these, designated AR1, ALl, AL2 and AL3, are located on DNA A, and the other two, designated BR1 and BLI, are on DNA B, where R and L indicate a rightward or leftward orientation with respect to the "common regions', intergenic regions of about 200 bp in each DNA and having an almost identical sequence. None of the protein products of these ORFs has been directly identified. We now present evidence which proves that ORF AR1 codes for the coat protein.TGMV was isolated from systemically infected Nicotiana benthamiana plants and purified by sucrose density gradient centrifugation as described by Stein et al. (1983). To ensure that the preparation was completely free from host proteins, virus was further purified by centrifugation for 20 h at 115 000 g in a caesium sulphate density gradient in a Beckman SW50.1 rotor. The virus formed a single band with a density of 1.34 g/ml. The ultraviolet absorbance spectrum of the virus had a maximum at 258 nm and a minimum at 235 nm with an A260 :A280 ratio of 1.56 (not corrected for light scattering). When the virus was disrupted by heating in 1 ~o SDS containing 1 ~ 2-mercaptoethanol for 3 rain at 100 °C and analysed by SDS-PAGE (Laemmli, 1970), followed by staining with Coomassie Brilliant Blue R250, a single protein band was detected.The Mr of the capsid polypeptide species was determined by mixing with protein standards (ovalbumin, 45K; deoxyribonuclease, 31K; trypsinogen, 24K; tobacco mosaic virus capsid polypeptide, 17-5K; cytochrome c, 12K), subjecting the mixture to SDS-PAGE in 12~, 14~, 16 ~o, 18 ~o and 20 ~ polyacrylamide gels and measuring the relative mobility (RF) of each protein t Present address:

show abstract

Starch-gel electrophoresis—Application to the classification of pituitary proteins and polypeptides

Cited by 980 publications

References 9 publications

The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

A periplasmic, α-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

Identification of the Coat Protein Gene of Tomato Golden Mosaic Virus

Contact Info

Product

Resources

About