The Release of Zinc From Carboxypeptidase and Its Replacement

Vallée, Bert L.; Rupley, John A.; Coombs, Thomas L.; Neurath, Hans

doi:10.1021/ja01550a094

Cited by 75 publications

(20 citation statements)

References 6 publications

(3 reference statements)

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“…16 9.1 ±0. 16 8.9±0.16 9.4±0.14 6.9±0.14 9.7±0.14 8.4+0.14 Thyrocalcitonin (-TCA or partially purified) was incubated in 0.1M ammonium carbonate, pH 8.8, or in 0.1M Tris, pH 7.5, with carboxypeptidase A (substrate:enzyme wt ratio, 20 to 30:1) at 25 or 37 C. Enzyme concentration was estimated from the absorbancy of the solution at 278 mju, assuming that an absorption of 1.94 was equivalent to 1.0 mg/ml (8).…”

Section: Methodsmentioning

confidence: 99%

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Tashjian

Warnock

1967

Endocrinology

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Porcine thyrocalcitonin was modified by enzymatic and chemical treatment for the purpose of studying the interrelationships between structure and hypocalcemic activity. Enzymatic treatment with trypsin, chymotrypsin, pepsin and polyphenyl oxidase led to rapid and complete inactivation of the hypocalcemic activity. We conclude from these experiments that thyrocalcitonin either is a simple polypeptide or contains a peptide moiety which is absolutely essential for hypocalcemic activity. Incubation of thyrocalcitonin in dilute HC1 or neutral Tris buffer at elevated temperature was accompanied by gradual loss of activity. Inactivation was also seen after treatment with hydrogen peroxide, photooxidation and treatment with n-bromosuccinimide, results demonstrating that there is at least one site in the thyrocalcitonin molecule that is sensitive to oxidation. The hypocalcemic activity which was lost during incubation with hydrogen peroxide could not be recovered by treatment of the oxidized products with cysteine, 2-mercaptoethanol or dithiothreitol. There were no detectable changes in hypocalcemic activity either after enzymatic treatment with carboxypeptidase A, leucine aminopeptidase and neuraminidase, or after chemical treatment with 2-OH-5-nitrobenzyl bromide, N-acetylimidazole, p-OHmercuribenzoate, iodoacetate and cyanogen bromide. Because thyrocalcitonin has not yet been purified, the sites of the modifications produced by various treatments cannot be shown. Results of experiments in which no loss of hypocalcemic activity was detected must be viewed with caution since it was not possible to show that amino acid residues in thyrocalcitonin were modified. However, these data may help to explain losses of hypocalcemic activity which occur during handling and to suggest new approaches to purification and characterization of thyrocalcitonin. (Endocrinology 81: 306, 1967)

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Section: Methodsmentioning

confidence: 99%

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Tashjian

Warnock

1967

Endocrinology

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“…Bovine carboxypeptidase A is a single polypeptide chain and contains one firmly bound zinc atom (74) essential for enzymatic activity (109). It splits only carboxyl-terminal residues from polypeptides containing an un blocked a-carboxyl group, and cleaves tripeptides about 103 times faster than the analogous dipeptides (110).…”

Section: Carboxypeptudase Amentioning

confidence: 99%

“…Previous chemical work had implicated the zinc (78,109) and one or two tyrosines (115,119). Al though no definite function could be assigned to the tyrosine, the zinc atom was thought to polarize the carbonyl group of the substrate (114,120).…”

Section: Mechanism Of Actionmentioning

confidence: 99%

X-Ray Diffraction Studies of Enzymes

Blow¹,

Steitz²

1970

Annu. Rev. Biochem.

200

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“…The use of cobalt also avoids the toxicity and cost of many conventional heavy-atom compounds. In addition, it has been demonstrated that cobalt can be substituted in metalloproteins for other metal ions that have similar binding geometry and coordination (Glusker, 1991;Harding, 2004;Maret & Vallee, 1993), such as zinc (Vallee et al, 1958;Vallee, 1973;Maret & Vallee, 1993;Hartwig, 2001;Ghering et al, 2004), iron (Sugiura et al, 1975) and copper (Calabrese et al, 1972). The proteins with the substituted metal often retain their native biological activity (Maret & Vallee, 1993).…”

Section: Introductionmentioning

confidence: 99%

The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

Gunčar

Wang

Forwood

et al. 2007

Acta Crystallogr F Struct Biol Cryst Commun

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Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using singlewavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 Å) is compatible with the Cu K wavelength (1.54 Å) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.

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The Release of Zinc From Carboxypeptidase and Its Replacement

Cited by 75 publications

References 6 publications

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

X-Ray Diffraction Studies of Enzymes

The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

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The Release of Zinc From Carboxypeptidase and Its Replacement

Cited by 75 publications

References 6 publications

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

X-Ray Diffraction Studies of Enzymes

The use of Co2+for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein

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The use of Co²⁺for crystallization and structure determination, using a conventional monochromatic X-ray source, of flax rust avirulence protein