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M A Wells

Publications and source records attributed to M A Wells.

At least 55 records · Page 3Linked to original sources
PubMed1995

Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Animals↗
PubMed1994

Hydration and localization of diacylglycerol in the insect lipoprotein lipophorin. A 13C-NMR study.

In order to probe the organization of diacylglycerol (DG) in lipophorin, 13C-enriched lipophorin was prepared for NMR investigations. We obtained 13C-enriched lipophorin labeled exclusively in DG by feeding insects tobacco leaves coated with [1-13C]palmitic acid or [1-13C]oleic acid. Lipophorins enriched up to 5% with a [13C]fatty acid were obtained by this procedure. NMR studies of the isolated lipophorin DG showed that palmitic acid accumulates almost entirely (> 90%) in the sn-1 position. Oleic acid was found equally distributed between the sn-1 and sn-2 positions, yielding a DG enriched equally at both positions. The 13C-NMR spectra of both [13C]palmitate- and [13C]oleate-enriched lipophorins showed that DG had one narrow carbonyl resonance indicative of rapid motion. A comparative analysis of the 13C carbonyl chemical shift data for DG in organic solvents, aqueous solutions, and dispersions with the DG carbonyl chemical shift of native lipophorin enriched in [13C]palmitate or [13C]oleate shows a high degree of water exclusion from the DG carbonyls in lipophorin. This result is consistent with the existence of a lipophorin lipid core containing most of the lipophorin DG. This study represents the first attempt to elucidate the organization of DG in lipophorin. The possibility of obtaining [13C]DG-enriched lipophorins, selectively enriched in one or both acyl chains of DG, should provide a powerful tool for further analysis of the organization and the dynamic properties of DG in native lipoproteins.

Animals↗
PubMed1994

Effect of diacylglycerol content on some physicochemical properties of the insect lipoprotein, lipophorin. Correlation with the binding of apolipophorin-III.

Diacylglycerol (DG) is the main lipid component of the insect lipoprotein lipophorin. In order to study the effect of DG content on the structure and properties of lipophorin and to analyze the role of DG in the binding of apolipophorin-III (apoLp-III), an exchangeable apolipoprotein, we developed a method that allows the modification of the DG content of lipoproteins. This method employs sn-1,2-dioctanoyl glycerol (diC8-DG). The degree of incorporation of diC8-DG was determined by including [14C]-diC8DG in the incubation and subsequent purification of the diC8-DG-loaded lipophorins in a KBr gradient. The efficiency of diC8-DG loading of lipophorin is time dependent, but high levels of loading are obtained in relatively short periods of time (100% in 3-4 h). For DG loading up to about 15% (w/w), the efficiency and rate of diC8-DG loading are independent of the presence of apoLp-III. DG loading above 15% (w/w) in the absence of apoLp-III resulted in aggregation of the particles. Lipophorin particles enriched up to about 30% with diC8-DG were obtained by this procedure. When lipophorin particles were loaded with diC8-DG in the presence of apoLp-III, it was observed that binding of apoLp-III was proportional to the amount of diC8-DG incorporated into the lipophorin particle, indicating that the only requirement for apoLp-III binding to lipophorin is an increased DG content. A decrease in the degree of order of the lipid phase of the lipoproteins was observed by anisotropy of fluorescence of diphenylhexatriene as the content of diC8-DG was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cation-promoted cyclic voltammetry of recombinant rat outer mitochondrial membrane cytochrome b5 at a gold electrode modified with beta-mercaptopropionic acid.

Reversible cyclic voltammetry of recombinant rat outer membrane (OM) cytochrome b5 was observed at a gold electrode modified with beta-mercaptopropionic acid. Electron transfer between the negative electrode surface and the negatively charged OM cytochrome b5 was promoted by the addition of divalent metal ions such as Mg2+ or Ca2+ and by the positively charged species poly-L-lysine. The titration of OM cytochrome b5 (0.1 mM) with poly-L-lysine resulted in a gradual positive shift of the E1/2 value which leveled off at +8 mV vs NHE when the poly-L-lysine:cytochrome b5 ratio reached a value of 2:1. Since the further addition of poly-L-lysine had no effect on the E1/2 value of the protein, it was concluded that a complex is formed in which two molecules of poly-L-lysine bind to each molecule of OM cytochrome b5. When the OM cytochrome b5-poly-L-lysine complex (0.1 mM) was titrated with Mg2+ or Ca2+ ions, the E1/2 value shifted gradually in the negative direction and leveled off at -40 mV vs NHE when the concentration of divalent ions reached 85 mM. When the voltammetric response of 0.1 mM cytochrome b5 was promoted by Mg2+ or Ca2+ ions, the minimum concentration of divalent cation necessary to produce a reversible voltammogram was 40 mM and the observed E1/2 was -46 mV vs NHE. On the other hand, only 0.2 mM [Cr(NH3)6]3+ was necessary to promote the reversible electrochemistry of 0.1 mM cytochrome b5. The half-wave potential observed under these conditions was -78 mV vs NHE. This indicates that there is a large dependence of the reduction potential of cytochromes b5 on the kind and concentration of multivalent ions in solution. A reduction potential of -102 mV vs NHE was obtained for OM cytochrome b5 (0.60 mM) by spectroelectrochemical titration in the presence of 0.4 mM [Ru(NH3)6]3+ and 1 mM methyl viologen, pH 7.0, mu = 0.1 M. This value is approximately 100 mV more negative than the reduction potentials reported for microsomal cytochromes b5 obtained from other sources under the same conditions. The binding interactions between OM cytochrome b5 and poly-L-lysine or Mg2+ ions were probed by investigating the isotropically shifted 1H NMR resonances arising from the heme in the OM cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Mercaptopropionic Acid↗
PubMed1994

Codon usage patterns among genes for lepidopteran hemolymph proteins.

Patterns in codon usage were examined for the coding regions of the 23 known lepidopteran hemolymph proteins. Coding triplets are GC rich at the third position and a significant linear relationship between GC content of silent and nonsilent (replacement) sites was demonstrated. Intron GC content was significantly lower than in coding regions and no relationship between intron GC content and the same at silent and nonsilent sites was found. Though hemolymph proteins are all produced by the same tissue--fat body--significantly less bias was observed when all moth sequences were pooled than when sequences of the two major species were analyzed separately, as predicted by the genome hypothesis. In cases where no statistically significant bias was observed, polar or acidic/basic amino acids were almost exclusively involved. Calculation of codon adaptation indices (CAI) was of limited value in quantifying the degree of codon bias and probably reflects the complexity of multicellular-organism life cycles and the changing patterns of gene expression over different developmental stages.

Animals↗
PubMed1994

Sequence of three cDNAs encoding an alkaline midgut trypsin from Manduca sexta.

We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.

Amino Acid Sequence↗
PubMed1994

Dietary control of late trypsin gene transcription in Aedes aegypti.

In Aedes aegypti the levels of midgut trypsin activity after feeding are directly proportional to the protein concentration in the meal. The mechanisms of this up-regulatory event were investigated by analyzing the expression of the late trypsin gene under different dietary conditions. Transcription of the gene was dependent on both the quality and quantity of protein in the meal. As measured by Northern blot analysis, the levels of late trypsin gene expression increased up to 100-fold 24 h after feeding on gamma-globulin, hemoglobin or albumin (100 mg/ml). In contrast, gelatin, histone, amino acids, saline or agarose were very poor inducers of transcription. The rates of late trypsin transcription induced during the first 24 h were directly proportional to the concentration of protein in the meal. These data further support the suggestion that the primary mechanism that regulates the synthesis of trypsin in the mosquito midgut is transcriptional regulation of the gene. This regulatory mechanism enables the midgut to maintain the appropriate balance between protease synthesis and the protein content of the meal.

Aedes↗
PubMed1994

Improving undergraduate biology education in a large research university.

The campus-wide Undergraduate Biology Research Program (UBRP) at the University of Arizona improves undergraduate science education by expanding student opportunities for independent research in faculty laboratories. Within the supportive community of a research laboratory, underclassmen, nonscience majors, and those aspiring to scientific careers all learn to appreciate the process of science. The Program impacts more than the students, promoting departmental cooperation, interdisciplinary collaborations, and improvements in undergraduate science education throughout a Research I University.

Adult↗
PubMed1994

Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta.

A triacylglycerol lipase, presumably the first enzyme involved in the mobilization of lipid from the insect fat body, has been purified to homogeneity from the fat body of Manduca sexta. The purification procedure involved polyethyleneglycol precipitation, and chromatography on DEAE-cellulose, phenyl-Sepharose, Q-Sepharose and hydroxylapatite. The final product, a protein with an M(r) = 76,000 by SDS-PAGE, was purified nearly 8000-fold from the original homogenate in a yield of about 11%. The enzyme catalyzed the hydrolysis of tri-, di-, and mono-oleoylglycerols, but showed highest affinity for tri- or dioleoylglycerol. Thus, under initial reaction conditions, the end products of trioleoylglycerol hydrolysis were: free fatty acids (66%), sn-2-monooleoylglycerol (24%), sn-1,2(2,3)-dioleoylglycerol (7%), and glycerol (3%). The fat body lipase exhibited a preference for hydrolyzing the primary ester bonds of acylglycerols, and did not show stereoselectivity toward either the sn-1 or sn-3 position of trioleoylglycerol. The enzyme had a pH optimum of 7.9, and was inhibited by diisopropylfluorophosphate, ATP, ADP, Mg2+, and NaF. The enzyme showed a strong tendency to aggregate, but was stable in detergent solutions at high concentration of glycerol. The polypeptide was phosphorylated by the cAMP-dependent protein kinase from bovine heart; however, phosphorylation did not cause activation of the enzyme. It is suggested that this fat body lipase could be analogous to the "hormone-sensitive lipase" of vertebrate adipose tissue.

Adenosine Diphosphate↗
PubMed1994

Exchangeable apolipoproteins of insects share a common structural motif.

Elucidation of the secondary structure of the exchangeable apolipoproteins has been hindered by the difficulty in producing crystals suitable for X-ray spectrographic analyses. Consequently, in order to analyze potential structure-function relationships in the family of insect exchangeable apolipoproteins, apolipophorins-III (apoLps-III), two apoLps-III cDNA clones, one from the palo verde beetle (Derobrachus geminatus) and one from the house cricket (Acheta domesticus), have been isolated and sequenced. Multiple sequence alignments of the deduced protein sequences with two previously reported apolipophorins-III from Manduca sexta and Locusta migratoria reveal low sequence identity, suggesting that these proteins are very old and are highly divergent. Computer-assisted predictions of protein structure and subsequent analyses, using the known secondary structure of Locusta migratoria apolipophorin-III as a control, indicate that these insect proteins are composed of five amphipathic helices with characteristics similar to those of the helical domains of the mammalian exchangeable apolipoproteins. Thus, although insect and vertebrate exchangeable apolipoproteins share a common function in assisting lipid transport, precise amino acid identity is less important than the common structural feature of multiple amphipathic helices. Moreover, because these proteins occur widely among insect species, even in those where flight is limited or absent, we hypothesize that apolipophorin-III has a more generalized function in lipid metabolism than had been previously proposed.

Amino Acid Sequence↗
PubMed1993

Mediterranean fruit fly, Ceratitis capitata (Wiedemann), mitochondrial DNA: genes and secondary structures for six t-RNAs.

The polymerase chain reaction was used to amplify six mitochondrial t-RNAs for Ala, Arg, Asn, Ser, Glu and Phe between genes for mitochondrial NADH dehydrogenases 3 and 5. With respect to Drosophila yakuba the gene order and direction of transcription is completely conserved. Analysis of secondary structure shows complete conservation of the anticodon loops but a number of differences in the dihydrouridine and T psi C loops with respect to Drosophila. However, differences are such that tertiary interactions that stabilize stacking are preserved. The use of the reported sequence in combination with PCR to explore population variability is discussed.

Animals↗
PubMed1993

Cloning and sequencing of the blood meal-induced late trypsin gene from the mosquito Aedes aegypti and characterization of the upstream regulatory region.

A 4.1 kb genomic clone of the late trypsin gene from the mosquito Aedes aegypti was isolated, mapped and subcloned. A 1.6 kb subclone, corresponding to 1.1 kb of upstream regulatory region and 0.5 kb of coding region, was sequenced. The gene has no introns within the coding region. The 5' end of the mature mRNA was mapped using primer extension analysis. A TATA box consensus sequence (TATAAA) was found at position -31 from the 5' end of the mature mRNA. A cluster of five repeat sequences homologous to the yeast GCN4 DNA binding site was found within 200 nucleotides upstream of the cap site. GCN4 is required for derepression mediated control of general amino acid biosynthesis in response to amino acid starvation in yeast. It activates the transcription of at least twenty different genes coding for enzymes involved in amino acid biosynthesis. The presence of this cluster of consensus sequences suggests that a protein similar to GCN4 might regulate expression of the late trypsin gene in the mosquito. Southern blot analysis of genomic DNA indicates that late trypsin is a single copy gene.

Aedes↗
PubMed1993

Polymorphic cDNAs encode for the methionine-rich storage protein from Manduca sexta.

By cDNA cloning and sequencing we have shown that Manduca sexta larvae produce three very closely related methionine-rich storage proteins, MMR1, MMR2 and MMR3. Out of 2256 nucleotides in the coding region, the cDNAs differ by at most twenty-one bases and this leads to a single amino acid difference between MMR1 and MMR2, and between MMR2 and MMR3, whereas MMR1 and MMR3 differ by two amino acids. Using both distance and parsimony methods, similarities between the M. sexta and Bombyx mori methionine-rich and arylphorin storage proteins were examined. Homologous proteins from the two species tend to be more closely related than are the two classes of storage proteins in a single species.

Amino Acid Sequence↗
PubMed1993

A comparison of three methods for isolating RNA from mosquitoes.

The two most common protocols for RNA isolation, Chirgwin et al., 1979, and Chomczynski & Sacchi, 1987, were compared with a protocol using glass powder. The quality of the recovered RNA was tested using agarose gels and Northern hybridization. With the glass powder protocol, we consistently obtained intact RNA, whereas with the other protocols we often obtained degraded RNA. Using specific radioactive probes, we measured ribosomal RNA and trypsin mRNA recovered from fed adult Aedes aegypti females. Our protocol gave a 40- and a 3-fold increase in recovery when compared with the Chirgwin and Chomczynski protocol, respectively. The method using glass powder is simple, fast, reproducible and gives a high yield of intact RNA.

Aedes↗
PubMed1993

p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition.

1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of 1-day old female honey bees, Apis mellifera L., were studied. 2. The calculated values obtained were Km = 2.27 x 10(-5)M and Vmax = 2.48 x 10(-8) mol/s per mg protein. 3. The inhibition mechanisms examined for four organophosphorus insecticides were highly competitive in nature and based on competitive inhibition coefficients the order of toxicity was naled > dichlorvos > cis-mevinphos = trans-mevinphos. 4. Comparisons are made with the alfalfa leafcutting bee, Megachile rotundata (Fab).

Animals↗
PubMed1992

Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.

Amino Acid Sequence↗
PubMed1992

Crystallization, structure determination and least-squares refinement to 1.75 A resolution of the fatty-acid-binding protein isolated from Manduca sexta L.

The molecular structure of an insect fatty-acid-binding protein isolated from Manduca sexta L. has been determined and refined to a nominal resolution of 1.75 A. Crystals used in the investigation were grown from 1.6 M-ammonium sulfate solutions buffered at pH 4.5 with 50 mM-sodium succinate, and belonged to space group P2(1) with unit cell dimensions of a = 27.5 A, b = 71.0 A, c = 28.7 A and beta = 90.8 degrees. An electron density map, phased with four heavy-atom derivatives and calculated to 2.5 A resolution, allowed for complete tracing of the 131 amino acid residue polypeptide chain. Subsequent least-squares refinement of the model reduced the R-factor from 46.0% to 17.3% using all measured X-ray data from 30.0 A to 1.75 A. Approximately 92% of the amino acid residues fall into classical secondary structural elements including ten strands of anti-parallel beta-pleated sheet, two alpha-helices, one type I turn, three type II turns, four type II' turns and one type III turn. As in other fatty-acid-binding proteins, the overall molecular architecture of the insect molecule consists of ten strands of anti-parallel beta-pleated sheet forming two layers that are nearly orthogonal to one another. A helix-turn-helix motif at the N-terminal portion of the protein flanks one side of the up-and-down beta-barrel. The functional group of the fatty acid is within hydrogen-bonding distance of Gln39, Tyr129, Arg127 and a sulfate molecule, while the aliphatic portion of the ligand is surrounded by hydrophobic amino acid residues lining the beta-barrel. The binding of the carboxylic acid portion of the ligand is very similar to that observed in P2 myelin protein and the murine adipocyte lipid-binding protein, but the positioning of the hydrocarbon tail after approximately C6 is completely different.

Animals↗