σ Complexes as biochemical and biophysical probes: 4. Development of a novel reporter group delivery system

A novel reporter group delivery system for the chemical modification of proteins and the investigation of enzyme mechanisms is proposed. The design of this system is based on structural analogy with pyridoxal phosphate and σ-complex adduct formation. Progress is described toward the synthesis of suitable compounds, via three different approaches involving structural modification of the pyridine nucleus. A number of new compounds have been prepared, and other directions for future investigations are indicated.     

A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney.

Pig kidney aldehyde reductase is inactivated by 2,3-butanedione, phenylglyoxal, methylglyoxal, and 1,2-cyclohexanedione. 2,3-Butanedione caused the most rapid loss in enzyme activity, the rate of loss being proportional to the concentration of 2,3-butanedione. Neither D-glyceraldehyde nor pyridine 3-aldehyde, both substrates for this broadly specific enzyme, protected the enzyme from inactivation but 1 mM NADPH or NADP completely prevented the loss of activity by 2,3-butanedione suggesting the involvement of arginine in the binding of cofactor. Nicotinamide mononucleotide (NMN) (reduced form) offered no protection to inactivation whereas ADP-ribose phosphate gave complete protection indicating that it is the latter portion of NADPH which interacts with the essential arginine. Both NMN and ADP-ribose phosphate are competitive inhibitors of aldehyde reductase with respect to NADPH. Butanedione-modified aldehyde reductase could still bind to a blue dextran-Sepharose 4B column suggesting that the modified arginine did not bind NADPH. This was confirmed by fluorescence spectra which showed that chemically modified aldehyde reductase caused the same blue shift of NADPH fluorescence as did native aldehyde reductase. Of additional interest was the quenching of NADPH fluorescence by aldehyde reductase which, with one exception, is in contrast to the fluorescence behavior of all other oxidoreductases.     

Compositional relatedness of aldehyde reductases from several species

Summary The amino acid compositions of several monomeric NADPH-dependent aldehyde reductases from a variety of species have been determined and analyzed by the difference index method of Metzger et al. (1968). The difference indexes among mammals range from 4.15 – 6.10 indicating considerable homology. Comparison of chicken aldehyde reductase with mammalian aldehyde reductases gave values in the range 6.8 – 9.9 suggesting a close relationship whereas the difference indexes for the enzymes from fruit fly and Baker's yeast versus vertebrate aldehyde reductases (10.9 – 14.4) indicate more distant relationships. The extent of sequence homology among aldehyde reductases from these species was estimated from a plot of difference index versus percent sequence difference for oxido-reductases of known sequence. From this plot, and using a mammal-chicken divergence time of 300 million years and a mammalian order split of 75 million years, the rate of evolution of aldehyde reductases was calculated to lie in the range 5.8 – 15.6% sequence difference per 100 million years. Comparison with rates of evolution of oligomeric dehydrogenases indicates that aldehyde reductases comprise the most rapidly evolving family of oxido-reductases. This is probably related to the monomericity of aldehyde reductases since there is a direct correlation between the number of subunits and the rate of evolution.     

Iatrogenic hyponatraemia of the newborn due to maternal fluid overload: a prospective study.

Over five weeks 136 out of 246 deliveries were studied. Maternal plasma sodium concentrations were normal at admission. At delivery no significant difference was found between maternal and infant cord plasma sodium concentrations. Twenty-four of the 41 mothers who had received only oral fluids during labour had infants whose cord plasma sodium concentrations were normal. Of the 95 mothers who had been given intravenous fluids, however, only 14 infants with normal plasma sodium concentrations, 31 had a concentrations of 130 mmol (mEq)/1 or less and nine of these had a concentration of 125 mmol/1 or less. There was a highly significant inverse relation between cord plasma sodium concentration and rate of fluid administration, suggesting that hyponatraemia was due to intravenous treatment with predominantly sodium-free solutions. Endogenous antidiuretic activity probably increases during labour, and synthetic oxytocin in large doses has been shown to have an antidiuretic effect. The dose used in this study did not appear to have such an effect. Glucose solutions are often used as a vehicle for oxytocin; 83% of all fluid intake in this study was 5% or 10% glucose in water. Fluid balance in labour should be supervised closely, and oxytocin should be given in a more concentrated solution.     

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Isolation and sequence of a peptide containing an essential lysine

Interaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with pyridoxal 5'-phosphate and sodium borohydride leads to inactivation and modification of two lysine residues per enzyme dimer that are thought to bind glucose 6-phosphate [Milhausen, M., & Levy, H.R. (1975) Eur. J. Biochem. 50, 453-461]. The amino acid sequence surrounding this lysine residue is reported. Following tryptic hydrolysis of the modified enzyme, two peptides, each containing one pyridoxyllysine residue, were purified to homogeneity and subjected to automated Edman degradation. The sequences revealed that one of these, a heptapeptide, was derived from the other, containing 11 amino acids. Supporting evidence for the role of the modified lysine is provided in the following paper [Haghighi, B., & Levy, H.R. (1982) Biochemistry (second paper of three in this issue)]. End-group analysis of the native enzyme revealed that valine is the N-terminal and glycine the C-terminal amino acid and provides support for the identity of the enzyme's two subunits.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Water utilization of tropical hardwood hammocks of the Lower Florida Keys

Summary Predawn water potential of representative plant species, together with stable isotope composition of stem water and potential water sources were investigated in four low-elevation tropical hardwood hammocks in the Lower Florida Keys, during a one year period. Hammock species had the lowest water potentials when soil water content was low and/or soil salinity was high, but differences in groundwater salinity had no effect on the water potential. Comparison of D/H ratio of plant stem water with soil and ground water corroborates the conclusion that they are primarily utilizing soil water and not groundwater. Thus, tropical hardwood hammocks are buffered from saline groundwater, and are able to thrive in areas where groundwater salinity is as high as 25. The effect of sea level rise on these forests may depend more on changes in the frequency of tidal inundation of the soil surface than on changes in groundwater salinity.     

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions.

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies.     

Organization of the gene for iso-rANP, a rat B-type natriuretic peptide

Using the polymerase chain reaction and oligonucleotide primers constructed from knowledge of the cDNA sequence we have sequenced the gene for iso-rANP, a peptide of the B-type of atrial natriuretic peptides. The overall organization of the rat iso-ANP gene is the same as that of ANP and BNP consisting of three exons and two introns at relatively similar positions. Iso-rANP and it's gene are more closely related to BNPs than ANP and yet there are significant differences at both the protein and DNA levels. Our results suggest that iso-rANP and BNP are distinct members of the same sub-family (B-type natriuretic peptides) within the family of natriuretic peptide genes.