Avidin. I. Isolation and characterization of the protein and nucleic acid

Fraenkel‐Conrat, H.; Snell, Neva; Ducay, Eufemio D.

doi:10.1016/0003-9861(52)90263-4

Cited by 67 publications

(15 citation statements)

References 22 publications

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“…0 M urea, the concentration at which the majority of the proteins lose their tertiary and quaternary structures [36], suggests that the enzyme under investigation retains considerable native structure under these conditions. Such a high degree of stability has been observed in the case of avidin [37] and streptavidin [38] in 6.0M urea. It has been suggested that this is due to the relatively weak denaturant property of urea [39, 401 compared to other chaotropic agents.…”

Section: Effect Of Denaturants On Native and Modified Enzymementioning

confidence: 86%

Bovine lens acylpeptide hydrolase

Sharma

Ortwerth

1993

European Journal of Biochemistry

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An acylpeptide hydrolase has been purified from bovine lens tissue by anion-exchange and hydrophobic-interaction chromatography. The enzyme, purified over 27 000-fold with 44% recovery, has a molecular mass of 300 kDa under native conditions. Under denaturing conditions it shows a subunit molecular mass of 75 kDa. The enzyme is inhibited by diisopropylfluorophosphate (iPr,P-F), phenylmethylsulfonyl fluoride and N-ethylmaleimide, indicating the presence of an essential serine residue and -SH group. Each subunit of the enzyme has one active serine residue which can be labelled with ['H]iPr,P-F. Nu-blocked amino acids in L form act as competitive inhibitors of the enzyme. The antibiotics penicillin-G and ampicillin partially inhibit the enzyme. Exposure of the purified enzyme to the proteases trypsin, chymotrypsin or elastase do not result in any loss of activity. Digestion of the native enzyme with bovine trypsin generates a 55-kDa protein containing the active-site serine and a 22-kDa polypeptide, indicating the presence of a unique trypsin site. Nterminal amino acid sequencing of the 55-kDa polypeptide shows that the bovine lens enzyme has a sequence at the trypsin cleavage site identical to the porcine liver acylpeptide hydrolase sequence 196-215. The data show that the split enzyme is as active as the native enzyme towards the synthetic substrate Ac-Ala-p-nitroanilide. The enzyme activity decreases with increasing urea, but 15% of the activity remains even in the presence of 6.0M urea. On removal of urea, complete recovery of the enzyme activity is observed. However, treatment with 1 M guanidinemC1 completely inactivates the enzyme.In recent years considerable interest has been shown in the enzymes involved in the acetylation of N-terminal amino acids in proteins and the removal of Ac-amino acid from proteins and peptides [l -71. Acylpeptide hydrolase belongs to a new class of serine-type peptidases which are relatively larger in molecular size [ 3 , 7-91 than members of the trypsin, subtilisin and carboxypeptidase Y families [lo]. Acylpeptide hydrolase has been isolated from rat liver [3, 81, bovine liver [9], porcine liver [8], rat brain [12], sheep erythrocytes [13], human erythrocytes [14, 151, rabbit muscle [16] and human placenta [17] . The enzyme from rat liver [3, 8,

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Section: Effect Of Denaturants On Native and Modified Enzymementioning

confidence: 86%

Bovine lens acylpeptide hydrolase

Sharma

Ortwerth

1993

European Journal of Biochemistry

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“…In fact, avidin concentration in the egg white (50 mg/ml; Awade, 1996) is well above the concentration levels used in this investigation. Indeed, early works have reported the isolation of avidin from the egg white in association with nucleic acids or with an acidic glycoprotein (Fraenkel-Conrat et al, 1952). These findings were later disregarded for the most part and considered the consequence of an artefact (Green, 1975).…”

Section: Discussionmentioning

confidence: 97%

“…Despite some evidence in early work for the existence of avidin-nucleic acid complexes in the egg white (Fraenkel-Conrat et al, 1952), subsequent reports consistently described these products as being a consequence of preparation artefacts, and the avidin-DNA interaction was described as non-specific. In the present work, we investigated the DNA-binding properties of avidin.…”

Section: Introductionmentioning

confidence: 88%

DNA condensation by high‐affinity interaction with avidin

Morpurgo

Radu

Bayer

et al. 2004

J of Molecular Recognition

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Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.

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“…With the use of azure A, which is approximately 3~ to 4 times as sensitive as the Schiff reagent (73), faint cytoplasmic staining at the theoretical limit of resolution of the light microscope was occasionally observed. There have been reports of the presence of large quantities (up to 500 times the amount in the nucleus) of cytoplasmic DNA in the chick embryo as well as in other eggs and embryos (4,16,17,20,25), but a positive Feulgen reaction generally has not been observed in the cytoplasm (cf. reference 76), and, indeed, it is often difficult or imposible to demonstrate a positive Feulgen reaction in some nuclei (6,70).…”

Section: Discussionmentioning

confidence: 99%

Intramitochondrial Fibers With Dna Characteristics

Nass

1963

The Journal of Cell Biology

288

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The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuelease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after I and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.

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Avidin. I. Isolation and characterization of the protein and nucleic acid

Cited by 67 publications

References 22 publications

Bovine lens acylpeptide hydrolase

Bovine lens acylpeptide hydrolase

DNA condensation by high‐affinity interaction with avidin

Intramitochondrial Fibers With Dna Characteristics

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