Shawn Doonan

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Abstract Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta- trans -2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4- 14 C] and MVA-[2- 14 C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[ 14 C] and terpinen-4-ol-[ 14 C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone ( trans -thujan-3-one), and preparations from flowerheads converted IPP-[4- 14 C] in 2·7% yield into geranyl and neryl β- d -glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.

Isopentenyl pyrophosphate isomerase from pig liver.

Abstract Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn2+ or Mg2+, the former being more effective. At the optimum pH and concentration of Mn2+, Km and V were 2.7 μ m and 6.7 μmol min−1 mg−1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.

The primary structure of aspartate aminotransferase from pig heart muscle determined in part using a protease with specificity for lysine

We have recently reported [1] partial amino acid sequences of the cytoplasmic aspartate aminotransferase from pig heart muscle, based on studies of peptides produced by digestion of the protein with pepsin and trypsin [2] and with thermolysin and elastase [ 1]. This work gave ten major fragments containing 395 amino acid residues. We now present the complete structure of the molecule which consists of a chain of 412 amino acid residues. The structure given here is essentially in agreement with that of Ovchinnikov et al. [3]. However, the authors gave no indication of the data which was used to overlap their previously reported 21 partial sequences [4]. We consider it important, for this reason and due to the large size of the molecule, to confirm the correctness of the published structure and to show how the structure was derived from our earlier data. An important tool used in the present work was a newly characterized protease isolated from the fruiting body of the basidiomycete Armillaria mellea [5]. The enzyme has been shown to be an endopeptidase cleaving proteins such as fibrinogen and casein N-terminal to lysine residues. Our results confirm this specificity and provide further information about the type of peptide bond hydrolysed by the enzyme.

Biosynthesis of irregular monoterpenes in extracts from higher plants

Abstract Extracts from Artemisia annua and Santolina chamaecyparissus converted 14 C-labelled IPP, DMAPP and DMVC into artemisia ketone, its corresponding alcohol, lavandulol and trans -chrysanthemyl alcohol with up to 12.0 % incorporation of tracer. DMVC was the most effective precursor under standard conditions and led to unequal distribution of tracer in the C-5 moieties. The same extracts interconverted cis and trans -chrysanthemyl alcohols and their pyrophosphates, artemisia ketone, and artemisyl alcohol in up to 10·4% yields, but geraniol, nerol and linalol or their pyrophosphates were not precursors of any of these compounds. Formation of artemisia ketone and its alcohol from C-5 intermediates was enhanced by NAD + and NADP + but was unaffected by absence of oxygen. These co-factors did not affect the yields of lavandulol or trans -chrysanthemyl alcohol. These observations suggest closely related biogenetic pathways to the three irregular skeltons that do not involve the usual C-10 intermediates of monoterpene biosynthesis: i.e. the biogenetic isoprene rule is not obeyed.

Homology of the primary structures of cytoplasmic and mitochondrial aspartate aminotransferases from pig heart

Aspartate aminotransferase (AAT) is one of a small group of enzymes which exist in different molecular forms in the cytoplasm and in the mitochondria of the same cell. The isozymes of AAT differ in electrophoretic and immunological properties [ 1 ] and in amino acid composition [2] ; they are, however, of similar molecular weight [2] . It is a matter of considerable interest whether the two forms of the enzyme are structurally related and arose by divergence from a common ancestral protein or represent a case of convergent evolution of function in originally unrelated proteins. A decision between these two possibilities has not previously been made for any pair of cytoplasmic and mitochondrial isozymes. We have recently reported the complete primary structure of cytoplasmic AAT from pig heart [3 ] ; similar results were obtained by Ovchinnikov et al. [4]. To establish the structural and evolutionary relationships between the cytoplasmic and mitochondrial isozymes, we are now carrying out studies of the amino acid sequence of the mitochondrial form. Results already obtained show clear homologies between the two structures.

The effect of sulfhydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro

Abstract When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p -hydroxymercuriphenyl sulfonic acid, but not N -ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N -Ethylmaleimide does not react with this group. N -Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.

A quantitative study of the subcellular distribution of aspartate aminotransferase isozymes in rat liver

Abstract 1. 1. Between 15 and 19% of the aspartate aminotransferase activity of rat liver is of cytosolic origin; the remainder is localised entirely in the mitochondria. 2. 2. Mitochondria do not contain detectable levels of the cytosolic isozyme or vice versa. 3. 3. In solutions of low ionic strength, damaged mitochondria release only small amounts of aspartate aminotransferase. but the enzyme is released quantitatively by exposure to high concentrations of salt.

Mechanisms of enzyme action

4.1. Chymotrypsin 4.1.1. The activation process 4.1.2. The mechanism of action of a-chymotrypsin 4.1.3. Protein side chains involved in the reaction 4.1.4. Results of X-ray diffraction analysis 4.1.4.1. Activation 4.1.4.2. Mechanism of action and catalysis 4.2. Other Serine Proteases 4.3. Carboxypeptidase-A 4.3.1. Activation of the zymogen 4.3.2. Chemical studies of the active site 4.3.3. Mechanism of action of the enzyme 4.3.4. Results of X-ray diffraction studies 4.4. Pancreatic Ribonuclease 4.4.1. Specificity and activity 4.4.2. Primary structure of bovine pancreatic ribonuclease 4.4.3. Chemical modification of the enzyme 4.4.4. The dependence of reaction rate on pH 4.4.5. A possible mechanism of action 4.4.6. Results of X-ray diffraction studies 4.5. Lysozyme 4.5.1. Results of X-ray diffraction analysis 4.5.2. The mechanism of action of lysozyme 4.5.3. Factors responsible for the catalysis 4.6. Enzymes Containing Cofactors and with Close Model System Analogies 4.6.1. Tryptophanase catalysed reactions 4.6.2. Transamination reactions 4.6.3. Decarboxylation of fl-ketoacids 4.6.3.1. Decarboxylation of acetoacetate 4.6.3.2. Decarboxylation of oxaloacetate

Specificity and Inhibition of Phosphatases in Tanacetum vulgare

Summary Cell-free extracts from Tanacetum vulgare contained acid and alkaline phosphatases and ATP-ases but no inorganic pyrophosphatase. p-Nitrophenyl phosphate and isopentenyl pyrophosphate were substrates but C10 and C15-pyrophosphates or acyl phosphates were unattacked. Michaelis-Menten kinetics were obeyed at the pH-optima. Fluoride was the most effective inhibitor and gave Ki values similar to those with phosphatases from other plants. However, in contrast to the previous situations the inhibition was uniformly competitive. Levels of acid phosphatase were significantly (ca. 2-fold) increased in extracts prepared from plants that had been stem-fed with glucose-1-phosphate or isopentenyl pyrophosphate. The levels in the unperturbed plant varied over the growing season in an out-of-phase manner with that of MVA-kinase. The significance of these observations for biosynthetic studies in vivo and to the choice of conditions for developing optimum cell-free systems that can sustain terpenoid synthesis is discussed.

Glycosidases in the submaxillary gland of the mouse: Sexual dimorphism and the effects of testosterone

Abstract 1. 1. The activities of a number of glycosidases in the submaxillary glands of the mouse and other animals were measured. 2. 2. Two enzymes, β- N -acetyl- d -glucosaminidase and α- d mannosidase, showed a sexual dimorphism in the mouse: higher levels were found in the male but values could be elevated in the female by administration of testosterone.