Denis C. Shaw

The enzymic cleavage of the carbon-phosphorus bond: Purification and properties of phosphonatase

Abstract 1. 1. Bacillus cereus degrades 2-aminoethylphosphonate by the following pathway: 2. 2. Reaction II above involves the enzymic cleavage of the carbon-phosphorus bond and has not been described previously. The enzyme responsible for this reaction has now been purified and studied; we have suggested that it be named “2-phosphonoacetaldehyde phosphonohydrolase”, and that the trivial name be “phosphonatase”. 3. 3. Phosphonatase shows optimal activity between pH 8 and 9, and has a K m for 2-phosphonoacetaldehyde of 40 μM. Apartfrom 2-phosphonoacetaldehyde, phosphonate shows virtually no activity with any of a number of phosphonates and phosphate esters tested. 4. 4. The active enzyme is composed of two similar subunits, each of an approximate molecular weight of 33 000–37 000. Mg2+ is required for activity, but this probably helps to maintain the enzyme in its aggregated state rather than play a catalytic role at the active site; no other metals have been shown to be necessary for activity. 5. 5. Phosphonatase is inhibited by CN−, S2− and SO32−, which suggests that disulphide bridges may be necessary in maintaining the tertiary structure of the activeform of the enzyme. 6. 6. Orthophosphite, which has been shown previously to abolish the growth of B. cereus on 2-aminoethylphosphonate, inhibits the activity of phosphonatase, but only in the presence of 2-phosphonoacetaldehyde or acetaldehyde. Experiments with [32P]orthophosphite showed that, although the inhibitor was bound tightly to phosphonatase, it did not form a covalent bond with the enzyme.

The tammar wallaby : A model to study putative autocrine-induced changes in milk composition

The marsupial newborn is immature and the mother has the capacity to alter milk composition significantly during lactation, presumably to meet the nutritional requirements of the developing young. Furthermore, macropodid marsupials may practice asynchronous concurrent lactation (ACL)7whereby the mother provides milk which differs in all the major components from adjacent mammary glands for two young of different ages. This phenomenon suggests that local regulation of mammary function, in addition to endocrine stimuli, is likely to be important for controlling milk composition. This paper explores the possibility that changes in sucking patterns of the young represent the first step in a mechanism to signal the mammary gland for putative autocrine-induced changes in milk composition.

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.

The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of thetWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

The purification and characterisation of chorismate mutase-prephenate dehydrogenase from Escherichia coli K12.

Abstract 1. 1.|Chorismate mutase-prephenate dehydrogenase was purified to homogeneity from Escherichia coli K12. 2. 2.|Some of the general properties of the enzyme including preliminary kinetic data and sensitivity to inhibition by tyrosine have been determined. 3. 3.|The molecular weight of the native enzyme was found to be about 82 000. 4. 4.|The amino acid composition and preliminary data on the nature of the sulphydryl groups were also obtained. 5. 5.|The significance of the results in relation to those obtained previously for the enzyme from Aerobacter aerogenes is discussed.

Genetics of lucilin, a storage protein from the sheep blowfly, Lucilia cuprina (Calliphoridae)

Lucilin, the main storage protein of larval fat body and hemolymph in the sheep blowfly, Lucilia cuprina, has been isolated as a series of trimers composed of subunits of 83,000±5% daltons. Extensive electrophoretically detectable polymorphism of lucilin subunit patterns occurs in wild and laboratory populations of Lucilia; from four to nine bands are seen in any one individual. Evidence from genetic, electrophoretic, immunological, and structural studies suggests the existence of a series of 12 or more closely related structural loci (designated Luc-1 to Luc-12) which may have arisen through gene duplication. Codominant allelic variation has been found at several of these loci. Luc-1 and Luc-3, and probably the other structural loci of the series, are located on chromosome 2.

Tyrosine biosynthesis in aerobacter aerogenes: Purification and properties of chorismate mutase-prephenate dehydrogenase

Abstract Chorismatemutase-prephenate dehydrogenase from extracts of Aerobacter aerogenes was purified to near homogeneity. The molecular weight of the enzyme was determined by gel filtration to be 76 000. The amino acid composition and some kinetic constants of the enzyme are reported. Titration of the sulphydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) revealed the presence of four sulphydryl groups in the protein, two of which are reactive towards DTNB only under denaturing conditions. Prephenate proptectsboth activities against inactivation by iodoacetamide.

Physicochemical characterization of lens proteins of the squid nototodarus gouldi and comparison with vertebrate crystallins

The main water-soluble proteins of squid lens (S-crystallins) have a molecular weight of 60 000, a sedimentation coefficient s020,w of 5.2 S, 20-30% alpha-helical secondary structure, and an unusually high methionine content (12%). The subunits of Mr 30 000 (major) and Mr 27 000 (minor) have related N-terminal amino acid sequences, but a very heterogeneous charge distribution with predominantly basic isoelectric points. Higher-Mr aggregates have similar secondary/tertiary structure and amino acid composition, but contain additional acidic subunits and Mr 35 000-40 000 subunits. S-crystallins resemble vertebrate beta-crystallins in their quaternary structure, and their N-terminal sequence shows analogy with the first 19 residues of calf beta/gamma-crystallin folding units. In the urea-soluble and urea-insoluble lens fractions polypeptides of Mr 58 000 and 80 000, respectively, predominate, which presumably correspond to the main cytoskeleton and membrane proteins. Water-soluble lens components of less than Mr 2000 were isolated which have ultraviolet absorption maxima at 327 and 370 nm.

Lactose Synthesis in a Monotreme, the Echidna (Tachyglossus aculeatus): Isolation and Amino Acid Sequence of Echidna α-Lactalbumin

alpha-Lactalbumin and lysozyme were each isolated from echidna (Tachyglossus aculeatus) milk by gel permeation and ion exchange chromatography. The alpha-lactalbumin modified the action of echidna milk galactosyltransferase to promote the synthesis of lactose but had very little effect on bovine galactosyltransferase. Echidna alpha-lactalbumin is a glycosylated protein with an apparent molecular weight of 20,000 (SDS-PAGE) whose concentration in the milk is very low compared with the concentrations of alpha-lactalbumin in the milk of other species. Its amino acid sequence is more similar to that of another monotreme, the platypus (Ornithorhynchus anatinus), than to the sequences of eutherian or marsupial alpha-lactalbumins. Echidna milk lysozyme, even at high concentrations, did not promote the synthesis of lactose by either echidna or bovine galactosyltransferase. We conclude that lactose synthesis in the echidna occurs by the same mechanism as that found in the platypus and other mammals.

Evolution of the Monotremes: Phylogenetic Relationship to Marsupials and Eutherians, and Estimation of Divergence Dates Based on α-Lactalbumin Amino Acid Sequences

The amino acid sequences of the α-lactalbumins of the echidna, Tachyglossus aculeatus, and the platypus, Ornithorhynchus anatinus, were compared with each other and with those of 13 eutherian and 3 marsupial species. Phylogenetic parsimony analyses, in which selected mammalian lysozymes were used as outgroups, yielded trees whose consensus indicated that the two monotremes are sister taxa to marsupials and eutherians and that the latter two clades are each others closest relatives. The data do not support the notion of a Marsupionta (monotreme–marsupial) clade. Pairwise comparison between the α-lactalbumins yielded maximum-likelihood distances from which divergence dates were estimated on the basis of three calibration points. The distance data support the view that the echidna and platypus lineages diverged from their last common ancestor at least 50 to 57 Ma (million years ago) and that monotremes diverged from marsupials and eutherian mammals about 163 to 186 Ma.