Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques.

The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism.     

1H NMR spectroscopic studies of calcium-binding proteins. 2. Histidine microenvironments in alpha- and beta-parvalbumins as determined by protonation and laser photochemically induced dynamic nuclear polarization effects

The microenvironments of the histidines in three isoforms of Ca(II)-bound parvalbumin (carp, pI = 4.25; pike, pI = 5.00; rat, pI = 5.50) have been examined with 1H NMR techniques to probe their protonation characteristics and photochemically induced dynamic nuclear polarizability (photo-CIDNP). The histidine at position 26 (or 25), present in all three of these proteins, shows absolutely no photo-CIDNP enhancement of its C2H or C5H resonances. Nor does this nonpolarizable histidine possess a normal pKa: values range only from 4.20 for carp to 4.32 for pike to 4.44 for rat. The C2H and C5H resonances of the histidine in this carp isoform split into doublets as the pH is lowered. The magnitude of this splitting depends on the magnetic field strength, temperature, and pH; however, the line intensities within each doublet are temperature-independent. Although the crystal structure of carp parvalbumin indicates that His-26 is exposed to solvent [Kretsinger, R. H., & Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326], we conclude that in solution this residue, in its unprotonated state, is part of the hydrophobic core of the protein. In contrast, His-48 in rat parvalbumin and His-106 in pike III parvalbumin show dramatic photo-CIDNP enhancements of their C2H, C5H, and beta-CH2 1H NMR resonances. Combined with its nearly normal pKa, 6.14, and exchange-broadened C2H resonance, the photo-CIDNP enhancement results for His-48 indicate that its microenvironment differs little from random-coil exposure, consistent with its presumed position on the solvent surface of helix C.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

19F nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Orientational order in the presence of a series of positional isomers of cis-octadecenoic acid

The 19F nuclear magnetic resonance (NMR) spectra of membranes of Acholeplasma laidlawii B enriched with one of a series of positional isomers of cis-octadecenoic acid plus small amounts of one of a number of isomers of monofluoropalmitic acid were interpreted in terms of an orientational order parameter (Smol). The variation of Smol with the position of the fluorine label in the liquid-crystalline state yielded an "order profile" with characteristics similar to those obtained via 2H NMR and which was relatively invariant regardless of the site of cis unsaturation. In the gel state, values of Smol approached the theoretical maximum, and the order profiles in the presence of different isomeric cis-octadecenoic acids displayed distinct dissimilarities. When the cis double bond was located proximal to the methyl terminus of the fatty acyl chain, a gradient of order across the bilayer was still evident in the gel state. When the cis double bond was located near the carbonyl head group, values of Smol were approximately equal at all chain positions. These observations were interpreted as indicating that in the gel state the stringency of packing restrictions is still subject to variation across the width of the bilayer. Relative overall orientational order among all isomers examined (specifically, 18:1c delta 4, delta 5, delta 6, delta 7, delta 8, delta 9, delta 10, delta 11, delta 12, delta 13, delta 14, and delta 15) varied directly as a function of proximity to the lipid gel to liquid-crystalline phase transition (Tm) (determined via differential scanning calorimetry) when compared at a constant temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.     

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Gel-state disorder in the presence of methyl iso- and anteiso-branched-chain substituents

The hydrocarbon chain orientational order parameters of membranes of Acholeplasma laidlawii B enriched with large quantities of a linear saturated, a methyl iso-branched, or a methyl anteiso-branched fatty acid plus small quantities of various isomeric monofluoropalmitic acid probes were determined via fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR) over a range of temperatures spanning the gel to liquid-crystalline phase transitions (determined by differential scanning calorimetry). Membrane orientational order profiles in the liquid-crystalline state were generally similar regardless of the particular fatty acyl structure, showing a region of relatively constant order preceding a region of progressive decline in order toward the methyl terminus of the acyl chain. In the gel state, the order profile of the linear saturated fatty acid enriched membranes was characteristically flat, with little head to tail gradation of order. In contrast, the methyl iso-branched and the methyl anteiso-branched enriched membranes exhibited a local disordering in the gel phase reflected in a very pronounced head to tail gradient of order, which remained at temperatures below the lipid phase transition. In addition, the methyl iso- and anteiso-branched fatty acid enriched membranes were overall more disordered than the membrane containing only linear saturated fatty acyl groups. Thus, at a constant value of reduced temperature below the lipid phase transition, overall order decreased in the progression 15:0 greater than 16:0i greater than 16:0ai, suggesting that these methyl-branched substituents lower the lipid phase transition by disrupting the gel phase lipid chain packing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyphenol oxidase produced during encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Affinity of chromatin constituents for isopeptidase

Isopeptidase is a novel eukaryotic enzyme that cleaves a structural chromatin protein, A24, stoichiometrically into H2A and ubiquitin. To understand the rapid turnover of ubiquitin in mitosis as wells as the high specific activity of the enzyme associated with metaphase chromosomes, attempts were made to determine chromatin constituents that show high affinity for this enzyme. Endogenous protease-free isopeptidase was prepared from calf thymus and applied to a Sepharose 4B affinity column on which histones, DNA, NHCP and ubiquitin were respectively immobilized. The enzyme proved to bind only histones. To further determine which of the histone fractions is involved, affinity columns with each histone fraction were also used. The enzyme showed affinity for all histone fractions. However, the strength of affinity varied in the order H2A>H³ H2B≥H4?H1, being inversely correlated with the ratio of basic/acidic amino acids in these molecules. These results suggest that the turnover of A24 in mitosis is controlled, at least in part, by the affinity of enzyme for histones, and also that such affinity is caused by a mechanism which cannot be explained simply by the electrostatic interaction between negatively charged enzyme molecules and positively charged histones.     

The vegetation of a New Zealand dune slack

The vegetation of a dune slack at Mason Bay, Stewart Island, New Zealand was found to comprise a mosaic of communities. Although the broad vegetational patterns could be correlated with the depth of the water table, the patterns were far from simple. Species diversity over the whole slack was lower than values reported from European dune slacks; even the most diverse communities did not reach European mean values.For nomenclature see Wilson in press. Vascular plants of Stewart Island. D.S.I.R., Wellington, New Zealand; Sainsbury (1955). A handbook of the New Zealand mosses. N.Z. R. Soc. 5: 1–490 & Hamlin (1972). Hepaticae of New Zealand, Dominion Museum, Wellington.