Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: A model system for analysis of structure-function relationships in the absence of subunit heterogeneity

Michael D. Brenowitz, Celia Bonaventura, Joseph Bonaventura

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein. In the absence of calcium ions, hexamers of Limulus IV are of higher oxygen affinity than monomers. Addition of calcium to the hexamers of Limulus IV hemocyanin results in cooperative oxygen binding. The cooperative oxygen binding of subunit IV hexamers is a clear demonstration that subunit heterogeneity is not a requirement for cooperativity in hemocyanins. The effects of monovalent and divalent cations on the function and self-assembly of Limulus IV hemocyanin are consistent with the proposal that this subunit is directly, involved in the calcium-dependent stabilization of the 48-subunit ensemble, which is a unique feature of horseshoe crab hemocyanin.

Original languageEnglish (US)
Pages (from-to)4707-4713
Number of pages7
JournalBiochemistry
Volume22
Issue number20
StatePublished - 1983
Externally publishedYes

Fingerprint

Horseshoe Crabs
Hemocyanin
Systems Analysis
Calcium
Oxygen
Monomers
Ionic strength
Osmolar Concentration
Self assembly
Molecules
Protons
Molluscs
Ions
Monovalent Cations
Proteins
Arthropods
Mollusca
Divalent Cations
Homopolymerization
Thermodynamics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assembly and calcium-induced cooperativity of Limulus IV hemocyanin : A model system for analysis of structure-function relationships in the absence of subunit heterogeneity. / Brenowitz, Michael D.; Bonaventura, Celia; Bonaventura, Joseph.

In: Biochemistry, Vol. 22, No. 20, 1983, p. 4707-4713.

Research output: Contribution to journalArticle

@article{1a369266847f440e84791a9e14e1d68f,
title = "Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: A model system for analysis of structure-function relationships in the absence of subunit heterogeneity",
abstract = "Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is {"}microheterogeneity{"} among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein. In the absence of calcium ions, hexamers of Limulus IV are of higher oxygen affinity than monomers. Addition of calcium to the hexamers of Limulus IV hemocyanin results in cooperative oxygen binding. The cooperative oxygen binding of subunit IV hexamers is a clear demonstration that subunit heterogeneity is not a requirement for cooperativity in hemocyanins. The effects of monovalent and divalent cations on the function and self-assembly of Limulus IV hemocyanin are consistent with the proposal that this subunit is directly, involved in the calcium-dependent stabilization of the 48-subunit ensemble, which is a unique feature of horseshoe crab hemocyanin.",
author = "Brenowitz, {Michael D.} and Celia Bonaventura and Joseph Bonaventura",
year = "1983",
language = "English (US)",
volume = "22",
pages = "4707--4713",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "20",

}

TY - JOUR

T1 - Assembly and calcium-induced cooperativity of Limulus IV hemocyanin

T2 - A model system for analysis of structure-function relationships in the absence of subunit heterogeneity

AU - Brenowitz, Michael D.

AU - Bonaventura, Celia

AU - Bonaventura, Joseph

PY - 1983

Y1 - 1983

N2 - Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein. In the absence of calcium ions, hexamers of Limulus IV are of higher oxygen affinity than monomers. Addition of calcium to the hexamers of Limulus IV hemocyanin results in cooperative oxygen binding. The cooperative oxygen binding of subunit IV hexamers is a clear demonstration that subunit heterogeneity is not a requirement for cooperativity in hemocyanins. The effects of monovalent and divalent cations on the function and self-assembly of Limulus IV hemocyanin are consistent with the proposal that this subunit is directly, involved in the calcium-dependent stabilization of the 48-subunit ensemble, which is a unique feature of horseshoe crab hemocyanin.

AB - Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein. In the absence of calcium ions, hexamers of Limulus IV are of higher oxygen affinity than monomers. Addition of calcium to the hexamers of Limulus IV hemocyanin results in cooperative oxygen binding. The cooperative oxygen binding of subunit IV hexamers is a clear demonstration that subunit heterogeneity is not a requirement for cooperativity in hemocyanins. The effects of monovalent and divalent cations on the function and self-assembly of Limulus IV hemocyanin are consistent with the proposal that this subunit is directly, involved in the calcium-dependent stabilization of the 48-subunit ensemble, which is a unique feature of horseshoe crab hemocyanin.

UR - http://www.scopus.com/inward/record.url?scp=0021113922&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021113922&partnerID=8YFLogxK

M3 - Article

C2 - 6626525

AN - SCOPUS:0021113922

VL - 22

SP - 4707

EP - 4713

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 20

ER -