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J H Law

Publications and source records attributed to J H Law.

At least 19 recordsLinked to original sources
PubMed1992

Lipophorin lipase from the yolk of Manduca sexta eggs: identification and partial characterization.

In the hawkmoth Manduca sexta high density lipophorin from adult insects (HDLp-A) delivers lipids to developing oocytes. During this lipid delivery HDLp-A is taken up by the oocyte and converted to a very high density lipophorin (VHDLp), which is stored in protein storage granules (yolk bodies). A membrane-free lysate of isolated M. sexta yolk bodies was demonstrated to contain lipoprotein lipase activity that hydrolyses the diacylglycerol of HDLp-A. With HDLp-A as a substrate yolk body lipophorin lipase (YBLpL) activity was shown to be maximal between pH 9 and pH 9.5. NaCl concentration was optimal between 0.7 M and 1 M. YBLpL activity required neither bovine serum albumin nor calcium ions but appeared to be stimulated by 5 mM EDTA. Diisopropyl fluorophosphate effectively inhibited YBLpL activity, indicating the presence of a serine in the active site of the enzyme. The identified lipase activity co-eluted with lipophorins and vitellins from the yolk in the void volume of a Sephadex G-75 gel filtration column. This observation suggests that the lipase has a Mr of more than 80,000, or that the enzyme is associated with the lipoproteins. Incubation of HDLp-A with yolk body lysate converted HDLp-A to two classes of higher density lipophorins. The highest density lipophorins produced during this incubation approached the density of VHDLp as it is isolated from mature eggs. The possible role of YBLpL activity in the delivery of lipids to developing oocytes is discussed.

Animals↗
PubMed1992

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.

Animals↗
PubMed1992

Preliminary biological characterization of a melanization stimulating factor (MSF) from the dorsal skin of the channel catfish, Ictalurus punctatus.

A two step fractionation of conditioned media made from the darkly pigmented dorsal skin of the channel catfish, Ictalurus punctatus, has produced fractions that contain a melanization stimulating factor (MSF). Isolated neural tubes of Xenopus laevis embryos exposed to conditioned media and to specific fractions exhibit greater melanization (increased numbers of melanized cells and elevated percentages of melanized cells), a greater number of dendrites per melanized cell, and a greater number of emigrated neural crest cells than control neural tubes. The presence of MSF activity in the darkly pigmented dorsal integument suggests a role for a molecule or molecules in the development and maintenance of the dorsal/ventral pigment pattern of this piscine species and possibly of other vertebrates.

Animals↗
PubMed1991

Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta.

A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific protein.

Amino Acid Sequence↗
PubMed1991

Molecular structure of an apolipoprotein determined at 2.5-A resolution.

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.

Amino Acid Sequence↗
PubMed1991

Structure-activity studies on adipokinetic hormones in Manduca sexta.

Structure-activity studies were performed for adipokinetic hormone (AKH) in Manduca sexta. Seven naturally occurring and four synthetic peptides of the red pigment concentrating hormone (RPCH)/AKH family were tested in larvae of M. sexta for activation of glycogen phosphorylase in fat body. pGlu at the N-terminal was found to be important for activity of peptides; however, Manduca AcGly1AKH is partially active. The amino acids at all positions appear to be of importance for activity, with the possible exception of the two serine residues in positions six and seven. Generally, the more amino acids are exchanged, the less the peptide will bind to the receptor. In M. sexta a beta-bend appears not to be important for the binding of peptides. Peptides ten amino acids long appear to be more active than shorter ones.

Amino Acid Sequence↗
PubMed1991

Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.

Animals↗
PubMed1991

An immunoreactive apoglycoprotein purified from Coccidioides immitis.

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and greater than 200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.

Antigens, Fungal↗
PubMed1991

Lipophorin structure analyzed by in vitro treatment with lipases.

Adult Manduca sexta high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I, -II, and -III) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-III. The amount of phospholipid remains constant in lipophorin (140 mol/mol of lipophorin), while the diacylglycerol content varies between different lipophorin species (310 mol/mol HDLp up to 1160 mol/mol LDLp). Both lipophorin particles were enzymatically depleted of phospholipid or diacylglycerol by in vitro incubation with either phospholipase A2 or triacylglycerol lipase. Albumin was used to remove free fatty acids generated during the reaction. Treatment with phospholipase A2 removed all phospholipids (except sphingomyelin) and the resulting particles were stable. Triacylglycerol lipase hydrolyzed large fractions of diacylglycerol. The resulting particles were smaller in size, higher in density, and devoid of apoLp-III. The particles retained apoLp-I and -II and the other lipid components, including a substantial amount of diacylglycerol. Structural integrity of diacylglycerol-depleted lipophorin was confirmed by electron microscopical analysis. When treated with both phospholipase A2 and triacylglycerol lipase, lipophorin precipitated. From these results we conclude that: 1) all phospholipid and apoLp-III are located at the surface of lipophorin, whereas diacylglycerol is partitioned between the sublayers and the surface of the particle; 2) both diacylglycerol and phospholipid play a role in stabilizing lipophorin in the aqueous medium; and 3) lipophorin can be extensively unloaded and still retain its basic structure, a necessary feature for its function as a reusable lipid shuttle.

Animals↗
PubMed1991

In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.

Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d approximately 1.109 g/ml) and by a low density lipophorin during flight (LDLp, d approximately 1.060 g/ml). Lipophorin presumably shuttles different lipids between sites of uptake or storage, and sites of utilization. In order to shuttle lipid, a lipid-depleted lipophorin should be able to reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d approximately 1.118 g/ml) or diacylglycerol (d approximately 1.187 g/ml) and subsequently radiolabeled in their protein moiety. Upon injection into adult moths, both particles shifted their density to that of native HDLp-A, indicating lipid loading. Also, upon subsequent injection of adipokinetic hormone, both particles shifted to a lower density (d approximately 1.060 g/ml) indicating diacylglycerol loading and conversion to LDLp. Both phospholipid and diacylglycerol loading were also studied using an in vitro system. The lipid-depleted particles were incubated with fat body that had been radiolabeled in either the phospholipid or the triacylglycerol fraction. Transfer of radiolabeled phospholipid and diacylglycerol from fat body to lipophorin was observed. During diacylglycerol loading, apoLp-III associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-III. The results show the ability of lipid-depleted lipophorins to reload with lipid and therefore reaffirm the role of lipophorin as a reusable lipid shuttle.

Animals↗
PubMed1990

Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins.

An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution.

Amino Acid Sequence↗
PubMed1990

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation.

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.

Animals↗
PubMed1989

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein.

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.

Animals↗
PubMed1989

The nucleotide sequence of a microvitellogenin encoding gene from the tobacco hornworm, Manduca sexta.

A microvitellogenin (mVg)-coding gene (mvg) has been isolated from a lambda phage library prepared from the tobacco hornworm, Manduca sexta. One of the lambda clones had a 15-kb insert and contained the entire mvg gene. A DNA fragment (3.0 kb) containing this mvg gene has been sequenced. Southern blot analysis showed that there may be more than one mvg gene in M. sexta. The putative transcriptional start point (tsp) for the cloned mvg was determined by primer extension analysis. This gene contains a single intron in the 5'-noncoding region. The 5'-flanking sequence was compared to the 5'-conserved regions of yolk polypeptide-encoding genes (yp) of Drosophila melanogaster. Two regions were found in the 5'-flanking sequence of the mvg gene that have 66% similarity to the D. melanogaster yp consensus sequence that is believed to be involved in gene expression controlled by ecdysteroids. Furthermore, the sequences flanking these two regions are also similar to the ecdysone-responsive elements found in several genes of D. melanogaster. In fact, preliminary experiments showed that mVg mRNA synthesis is induced by the 20-hydroxyecdysone. Four regions of the mvg gene resemble the upstream conserved regions of the two vitellogenin-encoding genes of the locust, Locusta migratoria. The nucleotide sequence of mvg has 70% similarity to the sequence of one or more of the 30-kDa hemolymph proteins of Bombyx mori. This indicates a very close evolutionary relationship between these proteins.

Amino Acid Sequence↗
PubMed1989

A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine.

The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.

Animals↗