1. Parvalbumin exists in two major forms, amounting to 2.5 and 3.2 g per kg of fresh muscle. 2. The composition divergence index between both forms, calculated from their amino acid composition, indicates 81% sequence identity; however both isoparvalbumins are immunologically distinct. 3. Beside Ca2+, perch parvalbumin binds 2 g atoms Mg2+ per mol with a dissociation constant of 10(-5) M. 4. Determination of the affinity for magnesium is of particular importance as there are indications that, in vivo, parvalbumin can remain in the Mg2+-state during the contraction-relaxation cycle instead of switching from the Ca2+-to the Mg2+-state and vice versa.

A solid-phase chelator for calcium was prepared by linking parvalbumin (a muscle calcium-binding protein of remarkable stability) to the polyacrylamide matrix of Bio-Gel P-60. The immobilized parvalbumin can be used repeatedly, and, due to its remarkable affinity for calcium, it is capable of lowering calcium concentration to ≤10−10m at neutrality. The affinity for calcium remains relatively high even at pHs as low as 4–5, at which complexants such as EDTA, EGTA, or Chelex would be quite inefficient. As immobilized parvalbumin binds Mg2+ with an apparent Kdiss 3.5 orders of magnitude higher than that of Ca2+, it can be used to control calcium concentrations even in the presence of magnesium. The affinity for calcium of any strong complexant can be determined by measuring Ca2+ distribution between this complexant and the solid-phase parvalbumin. Due to its outstanding affinity and selectivity for calcium, immobilized parvalbumin could prove to be a powerful tool in investigating the role of calcium in the regulation of metabolic processes.

Pure parvalbumins isolated from turtle, chicken, and rabbit white skeletal muscle have been characterized in terms of their physical, chemical, and immunological properties. As for the parvalbumins of most fish and amphibians, they have sedimentation constants S20,w of approximately 1.45 +/- 0.25 S and molecular weights of approximately 12,000, with little or no evidence for aggregation. They contain no tryptophan, at most one tyrosine, and a high proportion of phenylalanine, resulting in characteristic absorption spectra. All three parvalbumins contain 2 g atoms of calcium/mol bound with a KDiss less than or equal to 10(-6) M. Complete removal of calcium can be achieved by treatment with EDTA and EGTA or by a purified preparation of fragmented sarcoplasmic reticulum. By a direct analytical procedure, the concentration of parvalbumins in white skeletal muscle from the turtle, chicken, and rabbit was estimated at approximately 9 to 11, 0.2 to 0.4, and 0.6 to 1.1 g/kg, respectively. No parvalbumin or immunologically cross-reacting material could be detected in chicken white breast muscle, and very little was found in rabbit red muscle. All three proteins are immunologically distinct. A minor isoparvalbumin (approximately 2% of the major component) was found in turtle muscle only.

Parvalbumins, i.e. the low molecular weight, calciumbinding muscle proteins isolated heretofore exclusively from fish and amphibians, have been obtained in sizable amounts from the skeletal muscle of turtle, chicken, rabbit, and man. The finding that these proteins are not confined to lower vertebrates but have been conserved throughout evolution strongly suggests that they must possess a definite physiological function possibly related to the contractile process.

A stable and very sensitive reagent for the determination of zinc is obtained by removing zinc from pig kidney aminopeptidase, a commercially available metalloenzyme. Up to a given limit, the enzymatic activity of the reagent is strictly proportional to the concentration of zinc ions in the assay system. Aminopeptidase activity is determined by measuring the rate of release of p-nitroaniline from the chromogenic substrate L-leucine-p-nitroanilide. Thus, with a simple recording photometer, rapid and accurate determinations of free zinc ions in concentrations ranging from 5 pg-10 μg ml-1 can be achieved. The selectivity of the method is such that 0.1 ng Zn ml-1 can be assayed in the presence of a 10–100-fold molar amounts of a wide array of other cations with a relative error below 10%. The analytical procedure has been extended to the assay of Cu2+, Co2+ and Ni2+ in the ng ml-1 range.

1.1. Particulate aminopeptidase (EC 3.4.1.2) from pig kidney, which contains 2 gatoms of zinc per mole protein as the only metal component (Wacker et al. (1971) Helv. Chim. Acta 54, s473–485) is converted to an inactive metal-free apoaminopeptidase by electrodialysis, gel filtration, or treatment with chelating agents.2.2. Metal depletion does not significantly affect the molecular weight and electrophoretic mobility of the protein, but reduces the resistance of the enzyme towards denaturation under extreme conditions.3.3. Zn2+, Cu2+, Co2+ or Ni2+, but no other cations, restore activity to the apoaminopeptidase.4.4. Restoration of enzyme activity is directly proportional to the amount of divalent metal ions added up to 2 gatoms per mole of apoaminopeptidase; there is no increase of activity upon further addition.5.5. The data indicate that particulate aminopeptidase from pig kidney is a zinc metalloenzyme, and suggest that both atoms are essential for activity

AbstractPig kidney particulate aminopeptidase (EC 3.4.1.2) was purified by a modification of the procedure of Wachsmuth et al. [3] to a state of homogeneity according to criteria of ultracentrifugation and polyacrylamide gel electrophoresis, and some of its physical and chemical properties were determined.The purified enzyme (specific activity 30 ± 3 μmoles leucine‐p‐nitroanilide hydrolyzed/min/mg) has a s20,w = 9.82 S at pH 8 and a molecular weight of about 280,000 as determined by high speed sedimentation equilibrium. Divergence between the number‐, weight‐ and z‐ average molecular weights, and a sharp decrease of these values at low protein concentration, suggest that one is dealing with an associating‐dissociating system.Amino acid analysis revealed the presence of considerable quantities of carbohydrates in the enzyme. Colorimetric, gas chromatographic and enzymatic analyses demonstrated the presence of glucosamine, galactose, mannose, fucose and sialic acid residues in the ratio of 1:0.89:0.75: 0.13: 0.13, amounting to ca. 400 residues or 20% (56,000 daltons) of the molecular weight. With the exception of sialic acid, the carbohydrate content was remarkably constant from preparation to preparation.Analyses by both atomic absorption spectrometry and the dithizone method showed that zinc–the only metal found in significant amount–was always present in the ratio of 2 atoms per molecule. It is therefore proposed that pig kidney particulate aminopeptidase is a Zn‐containing glycoportein.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAlpha-Amylases as Calcium-Metalloenzymes. I. Preparation of Calcium-free Apoamylases by Chelation and Electrodialysis*Eric A. Stein, Julia Hsiu, and Edmond H. FischerCite this: Biochemistry 1964, 3, 1, 56–61Publication Date (Print):January 1, 1964Publication History Published online1 May 2002Published inissue 1 January 1964https://doi.org/10.1021/bi00889a010RIGHTS & PERMISSIONSArticle Views173Altmetric-Citations70LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAlpha-Amylases as Calcium-Metalloenzymes. II. Calcium and the Catalytic Activity*Julia Hsiu, Edmond H. Fischer, and Eric A. SteinCite this: Biochemistry 1964, 3, 1, 61–66Publication Date (Print):January 1, 1964Publication History Published online1 May 2002Published inissue 1 January 1964https://doi.org/10.1021/bi00889a011Request reuse permissions Article Views579Altmetric-Citations109LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (791 KB) Get e-Alertsclose Get e-Alerts

1.1. Crystalline Bacillus subtilis α-amylase, which has been reported to be a calcium metalloenzyme, also contains small amounts of zinc. Under normal conditions, this enzyme exists in the form of a dimer.2.2. Dissociation of the dimer takes place in the presence of metal-binding agents whereas dimerization is induced by zinc ions. One atom of zinc per molecule of dimer is taken up by the protein during dimerization and released as dissociation proceeds according to the following scheme: [Prot—Cax—Zn—[Prot—Cax]2[Prot—Cax]+Zn3.3. Formation of this zinc-protein complex appears to be peculiar to B. subtilis α-amylase. Intermolecular cross-linking under the influence of zinc has not been observed with α-amylases from human saliva, hog pancreas, or Aspergillus oryzae.4.4. Calcium ions do not induce dimerization of bacterial amylase.

AbstractThe properties of two crystalline α‐amylases obtained from two different strains of B. subtilis are described. The pH optima, stabilities, temperature coefficients, absorption spectra, sedimentation and electrophoretic mobilities have been determined for both enzymes and found to be identical.