University of Cambridge

Date:
1968
Reference:
PP/CRI/E/1/16/7
Catalogue details

Licence: In copyright

Credit: University of Cambridge. Source: Wellcome Collection.

    7/19
    The amino acid sequence of haemerythrin has been completely established. This provides a basis for reasoned speculation as to groups involved in subunit interactions and in conformational changes in presence of various additives. SUBUNIT ARRANGEMENTS: THE ELECTRON MICROSCOPE PICTURE R.C. Valentine, National Institute for Medical Research, Mill Hill, London N.W.7., England. Protein molecules scatter too small a part of the electron beam to be seen directly by electron microscopy. However, dense negative stains can be used to outline the molecules and, in favourable cases, the details of subunit structure are shown. Since oligomeric protein molecules assemble themselves by the spontaneous aggregation of their subunits, they almost certainly have point-group symmetry. The possible types of symmetry are limited and this simplifies the interpretation of the electron micrographs. A model which lacks suitable symmetry is likely to be wrong. The various types of symmetry that have been seen will be illustrated. There are now a sufficient number of protein molecules whose subunit arrangement is reasonably well established for some generalisations to be possible. Most significant perhaps is the rarity of the simplest type of point-group symmetry, that of the cyclic ring. In general oligmeric proteins are either dimers or have dihedral symmetry (i.e. are cyclic rings of dimers). Exceptions are a few large molecules with cubic symmetry. The picture of oligomeric proteins may suggest how they have evolved. Cyclic symmetry demands an acceptor-donor type of bond between the subunits (heterologous) which could be provided for example by hydrogen bonding or ionic links. Such bonds are possibly not suited for initiating polymerisation since they are directional and weak, especially in salt solutions. Thus several such bonds may be simultaneously required to hold the units together, which can only occur when the topography of the opposing surfaces is ideally arranged. On the other hand, hydrophobic bonding is less directional and allows a degree of rotation to give the best fit between the surfaces. In addition it brings like surfaces together and the polymerisation is limited to forming dimers. These have a two-fold axis of rotational symmetry with the possibility of any bond being doubled in the symmetrical position on the other side of the axis (isologous bonding). In several cases there is evidence that the subunits of the dimer are similar but not identical. The molecule is then controlled by two genes, which may allow a more rapid evolution of a satisfactory bond. Dimers may further polymerise to tetramers, where two additional types of isologous bonds are possible, or to dihedral rings. These rings involve additional bond only and this can be heterologous. It is stabilised by being effectively doubled by the stronger dimer bond. Proteins composed of two very different types of subunit provide an interesting sophistication. Relationship between Subunit Structure and the Catalytic Activity of Escherichia coli Glutamine Synthetase E.R. Stadtman, Bennett M. Shapiro and Ann Ginsburg, National Heart Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Cellular regulation of glutamine synthetase activity in E^ coli is achieved by 4 separate mechanisms: (1) Repression of enzyme synthesis by high levels of ammonium ion; (2) feedback inhibition by each of eight different end products of glutamine metabolism; (3) modulation of feed back inhibition and divalent ion specificity by co-valent attachment of adenylyl groups in phosphodiester linkage to tyrosine residues on the enzyme. Hydrodynamic measurements and chemical data on homogeneous preparations show that the E^_ coli glutamine synthetase has a molecular weight of 600,000 and is composed of 12 apparently identical subunits. Examination in the electron microscope (by R.C. Valentine) show that the subunits are arranged in two hexagonal layers. As isolated, the enzyme (so-called taut form) contains up to 40 equivalents of bound Mn++ and is very resistant to reaction with sulfhydryl reagents or to denaturation by mild treatments; viz 1 M urea, or alkaline pH. With removal of the Mn++, the enzyme is converted to a relaxed form which is indistinguishable from the taut form, with respect to molecular weight and subunit structure. However relaxation is accompanied (a) by a loss in catalytic activity, (b) by exposure of 4 sulfhydryl groups per subunit (to reaction with organic mercurials or alkylating agents) (c) by the transfer of 1-2 aromatic amino acid residues per subunit from a non-polar to a more polar environment and (d) by a marked increase in susceptibility to complete dissociation of subunits by treatment with 1 M urea. With addition of Mg++ or Mn++ the relaxed enzyme is converted to a Tightened form that is essentially identical with the original taut enzyme except, as shown by electron microscopy (B. Shapiro and R.C. Valentine), the double hexagon molecules have a marked tendancy to undergo face to face interactions producing long hexagonal tubes which in turn undergo colinear aggregation and precipitate from solution at low protein concentrations. Treatment of the relaxed enzyme with 1M urea leads to complete dissociation of the subunits. With addition of Mn++ the subunits reassociate to form ill-defined double hexagon aggregates that are transiently catalytically active; with time, these undergo degeneration and complete loss of catalytic activity. The glutamine synthetase isolated from bacterial cells grown under conditions of nitrogen excess contain up to 12 equivalents (i.e., one per subunit) of co-valently bound adenylic acid residues. These are bound in phosphodiester linkage to a particular tyrosine residue on the enzyme. In contrast, glutamine synthetase isolated from bacteria grown under conditions of nitrogen starvation possesses few if any covalently bound AMP groups. E. coli contains an adenylyl transferase, which in the presence of ATP, glutamine and unadenylylated glutamine synthetase, catalyzes adenylylation of the enzyme. This appears to be identical with the glutamine synthetase inactivating enzyme previously described by H. Holzer and his associates.
    digitised image 1
    digitised image 2
    digitised image 3