Synthesis and properties of new coenzyme mimics based on the artificial coenzyme CL4.

We have previously shown that a range of nicotinamide containing 'biomimetic coenzymes' function as active analogues of NAD+ in the oxidation of alcohols by horse liver alcohol dehydrogenase (HLADH), despite their apparently astonishing lack of structural similarity to the natural coenzyme. The simplest structure as yet shown to exhibit activity is the biomimetic coenzyme CL4. To investigate the effect of the structure of this truncated artificial coenzyme on its activity, a range of close structural analogues of CL4 were designed, synthesized and characterized. The electrochemical reduction potentials of the analogues were strongly influenced by the nature of the groups attached to the pyridine ring. All of the analogues could be chemically reduced using sodium borohydride, to give compounds with altered UV-visible absorption and fluorescence properties. An HPLC-based assay suggested that two of the new analogues were coenzymically active in the oxidation of butan-1-ol by HLADH, with one displaying a significantly higher activity than CL4. The results demonstrate which features of the structures of the coenzymes lead to desirable electrochemical and spectroscopic properties, but suggest that the structural requirements for a functional coenzyme are quite stringent. These observations may be used to design an artificial coenzyme which combines the best features of those studied so far.

Expression of snowshoe hare bunyavirus S RNA coding proteins by recombinant baculoviruses.

Recombinant baculoviruses have been constructed that express the two snowshoe hare (SSH) bunyavirus proteins coded in overlapping reading frames of the SSH S viral-complementary RNA species (namely the nucleoprotein, N, and the nonstructural protein, NSS). The 26.5 kDa N protein, which is read from the first AUG of the mRNA containing the SSH S sequence, was expressed at a high level (estimated to be ca 40% of the stained cellular proteins in recombinant baculovirus-infected Spodoptera frugiperda cells). This level of expression was much higher than that of the 10.5 kDa NSS protein made at the same time (estimated to be less than 1% of the stained proteins), presumably due in part to lower levels of translation initiation from the second AUG (19 nucleotides downstream). Bal31 nuclease digestion was used to delete the first ATG of the SSH DNA sequence in the baculovirus transfer vector and BamHI was used to remove downstream N coding sequences. A second recombinant baculovirus was constructed from the products that only expressed the SSH NSS protein. The yield of NSS protein was estimated to be of the order of ca 2% of the stained cellular proteins. A third recombinant transfer vector made from the products of the Bal31 digestion, fortuitously possessed a new ATG 8-10 nucleotides upstream of the NSS ATG. A recombinant virus derived from this vector synthesized essentially similar quantities (ca 2% each) of both the NSS protein and a 16.7 kDa N-related product.

Measurement of surface charge of baculovirus polyhedra.

The isoelectric points of three baculoviruses, Trichoplusia ni nuclear polyhedrosis virus (NPV), T. ni granulosis virus, and Spodoptera littoralis NPV were identified by cell electrophoresis. At neutral pH polyhedra were negatively charged. T. ni NPV polyhedra were reacted with a number of reagents which could potentially attach to or degrade their surface structure. This gave information on the components that contribute to the charge profile of T. ni NPV. This is discussed in relation to the use of polyhedra as biological control agents against insect pests.

Hydrophobic interactions involved in attachment of a baculovirus to hydrophobic surfaces.

The hydrophobic interactions of Trichoplusia ni nuclear polyhedrosis virus were characterized by hydrophobic interaction chromatography. The determination of the hydrophobic force and some of the factors that influence its size is discussed in relation to the attachment to leaf surfaces of polyhedra during their use as biological control agents against insect pests.

A new rapid procedure for the preparation of plasmid DNA.

This report describes a simple and efficient procedure for the isolation of plasmid DNA free from chromosomal DNA, cellular RNA, and protein. The technique comprises a modified cleared lysate procedure of D.B. Clewell and D.R. Helinski (1969, Proc. Natl. Acad. Sci. USA, 62, 1159-1166) followed by high-performance liquid chromatography on a Dupont Bioseries GF250 surface stable diol-coated silica gel permeation column (Zorbax) for the final purification of the plasmid DNA. The use of HPLC facilitates rapid and high-resolution separations within 3-4 h. Plasmid DNA produced in this manner retains its biological activity and exhibits yields equal to those obtained by the conventional cesium chloride-ethidium bromide density centrifugation method.

Preparative high-performance liquid affinity chromatography.

The reactive triazine dye, Procion Blue MX-R, has been covalently attached to preparative-grade silica and used for the large-scale purification of rabbit muscle lactate dehydrogenase by high-performance liquid affinity chromatography (HPLAC). Purified dye was coupled directly to glycol-silylated silica via the reactive triazine ring to yield an adsorbent containing 12 mumol dye/g silica. Essentially homogeneous lactate dehydrogenase in 80% overall yield was obtained from crude extracts. Thus this report demonstrates the potential for adapting the speed of operation and resolution shown for triazine dye-HPLAC in analytical applications to preparative protein purification.

Affinity labelling of enzymes with triazine dyes. Isolation of a peptide in the catalytic domain of horse-liver alcohol dehydrogenase using Procion blue MX-R as a structural probe.

Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion blue MX-R, a dichlorotriazinyl structural analogue of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37 degrees C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 microM. Methoxylated Procion blue MX-R does not inactivate the enzyme. The inactivation of horse liver alcohol dehydrogenase by Procion blue MX-R is competitively inhibited by the pyridine nucleotides NAD+ and NADH. Quantitatively inhibited horse liver alcohol dehydrogenase contains 1 mol dye/mol subunit of Mr 40 000. Chymotryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a single blue peptide which on sequencing and analysis yields an amino acid sequence of: (formula; see text) with the affinity label, Procion blue MX-R, unambiguously identified as being attached to the thiol side chain of Cys-174 in the catalytic domain of the enzyme. The specific active-site-directed reaction of Procion blue MX-R with horse liver alcohol dehydrogenase is interpreted in terms of the known crystallographic structure of the enzyme.

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation.

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically 'frozen' during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca2+ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromatography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18--20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3--0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 microM epinephrine.