Robert O. Ryan

Arylphorin from Manduca sexta: Carbohydrate structure and immunological studies

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.

Lipid transfer protein from Manducasexta hemolymph

A hemolymph lipid transfer protein (LTP) was isolated from the tobacco hornworm, Manduca sexta. LTP catalyzes net lipid transfer between isolated hemolymph lipoproteins in vitro. An isolation procedure employing density gradient ultracentrifugation and gel permeation chromatography produced a purified protein. LTP is a very high density lipoprotein with a particle Mr greater than 500,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that LTP is comprised of two apoproteins: apoLTP-I (Mr approximately 320,000) and apoLTP-II (Mr approximately 85,000). LTP may have a physiological role in altering the lipid content and composition of the major hemolymph lipoprotein, lipophorin.

A novel procedure for the purification of apolipophorin III

Abstract A procedure for the purification of the lipophorin apoprotein, apolipophorin III (apoLp-III), is described. Apolipophorin III was isolated from both Manduca sexta and Thasus acutangulus lipophorins. Lipophorin was delipidated by extraction with chloroform-methanol, the apoLp-III extracted from the residual apoproteins and contaminating apoproteins removed by chromatography on concanavalin A sepharose. The isolated M. sexta apoLp-III was identical to apoLp-III isolated by Kawooya et al. (1984) ( J. biol. Chem. 259 , 10,733–10,737). T. acutangulus apoLp-III was determined to be a non-glycosylated polypeptide of M r = 20,000. Amino acid analysis revealed that the composition of T. acutangulus apoLp-III was similar to that of M. sexta apoLp-III. Both apoproteins lack cysteine and tryptophan and possess one or two tyrosines, which accounts for the observed A 230 A 280 ratio of ten or more. The biological activity of apoLp-III, prepared by this method, was assessed by the ability of radioiodinated apoLp-III to associate with their respective host lipophorin in vivo .

Arylphorin from the haemolymph of the larval honeybee, Apis mellifera

Abstract Manduca sexta anti-arylphorin serum was used to detect a cross-reactive protein in the haemolymph of fifth instar Apis mellifera larvae. The honeybee antigen was purified by preparative density gradient ultracentrifugation, gel permeation and ion exchange chromatography. The purified protein ( M r ⋍470,000 ) has a hexameric subunit composition ( M r ⋍74,000 ). The protein has a high percentage of aromatic amino acids, possesses covalently bound carbohydrate and non-covalent lipid. This protein appears to meet all the criteria of the insect serum proteins designated arylphorins and is the first example of this class to be isolated from Hymenoptera.

Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.

Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-high-density lipoproteins

Facilitated diacylglycerol exchange between Manduca sexta [3H]diacylglycerol-labeled high-density lipophorin and Heliothis zea very-high-density chromolipoprotein was studied. M. sexta lipid transfer particle was employed in assays which measured exchange of [3H]diacylglycerol. Following incubations with lipid transfer particle, donor and acceptor lipoproteins were reisolated by density-gradient ultracentrifugation to determine facilitated exchange. The reaction was limited to diacylglycerol exchange, while donor or acceptor particle apoprotein exchange did not occur. Lipid analysis of donor and acceptor lipoproteins after the lipid-exchange reaction revealed that the labeled diacylglycerol remained unchanged. Lipid transfer particle-catalyzed diacylglycerol exchange was linear up to 0.3 micrograms lipid transfer particle protein in a standard assay and exchange occurred at a rate of 2.5 micrograms diacylglycerol min-1.micrograms-1 lipid transfer particle protein. The assay method was used to show that the hemolymph concentration of lipid transfer particle increased during development.

Lipophorin from the grasshopper, Gastrimargus africanus: Isolation and properties of apolipophorin III

The major hemolymph lipoprotein, lipophorin, was isolated from the grasshopper, Gastrimargus africanus. In the resting form the lipoprotein is composed of two apoproteins, apolipophorin I (Mr 250,000) and apolipophorin II (Mr 80,000). Following the administration of synthetic locust adipokinetic hormone to adult G. africanus the density of lipophorin decreased from 1.10 to 1.05 g/ml, and a third apoprotein, apolipophorin III (apoLp-III), associated with the particle. ApoLp-III was isolated from the low density form of lipophorin, using organic solvents to extract the lipids and to denature apolipophorin I and II. The purified apoLp-III has an Mr = 20,000 and contains 5.3% carbohydrate. The protein and its physiological functions appear similar to apoLp-III of Manduca sexta, although the lepidopteran apolipoprotein has a lower molecular weight and does not contain carbohydrate.