Private consultant and owner of “Robotics Ultra-High Through-put Biologics Purification, LLC.” West Hollywood, CA. Protein biochemist with 25 years of expertise in research and development of therapeutic biologics.
The hemolymph of adult Munducu aextu (tobacco hornworm) contains a 17,000-dalton protein that can... more The hemolymph of adult Munducu aextu (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin 111. Apolipophorin I11 was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin I11 was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin I11 is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr *This study was supported by Grant 29238 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Two cercaria emitted from the snail Melanoides tuberculata are illustrated, one of which encysts ... more Two cercaria emitted from the snail Melanoides tuberculata are illustrated, one of which encysts on the gills of the fish Tilapia zilli. The parasite is shown to have a marked preference for the anterior gills and especially the middle part of each gill. Comparisons between the respiration and ventilation rates of experimentally infected and uninfected fish were made at two levels of activity. At the slower flow rate the parasitized fish have significantly higher oxygen consumptions than do the uninfected controls.
larval stage of the cotton leaf-worm Spodoptera littoralis (Boisd.) (Noctuidae, Lepidoptera) in A... more larval stage of the cotton leaf-worm Spodoptera littoralis (Boisd.) (Noctuidae, Lepidoptera) in Alexandria region. 2. ang. Ent. 74, 332-336. HOSNEY, M. M.; KOTBY, F. A., 1960: Host plants favoured by cotton leafworm moth, Prodenia litura F., for egg-laying, and their value as egg-mass trap crop. Bull. SOC. Ent. Egypte 44, 223-234.
The effect of different factors, such as stage of development, age, sex and pregnancy, on zymogra... more The effect of different factors, such as stage of development, age, sex and pregnancy, on zymograms of the tsetse fly, was studied for eight different enzyme systems. The study provided information with respect to the collection of material in the field and subsequent storage and transport to the laboratory.In a laboratory strain of G. morsitans, genetic polymorphism was demonstrated for five different gene loci, involved in the control of leucine‐amino peptidase, malic enzyme, and alkaline phosphatase. Several allozymes were found to occur at the lap3 locus and at the me‐locus.RésuméVARIATION GENETIQUE EN DIVERS SYSTEMES ENZYMATIQUES DANS LA MOUCHE TSETSE GLOSSINA MORSITANSL'effet de différents facteurs comme le stade de développement, l'âge, le sexe et l'état gravide sur les zymogrammes de la mouche Tsétsé a été étudié pour divers systèmes enzymatiques. Cette étude donne des informations en relations avec le matériel récolté sur le terrain et aussi après le stockage et...
The catalytic domain of human tumor necrosis factor- (TNF-) converting enzyme (TACE) was expresse... more The catalytic domain of human tumor necrosis factor- (TNF-) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF- to generate the mature TNF- in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF--specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative effica...
sity gradient ultracentrifugation in the course of the loading process indicates that apoLp-I11 i... more sity gradient ultracentrifugation in the course of the loading process indicates that apoLp-I11 is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-111, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-I11 adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-111 subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-I11 showed that the two apoLp-I11 molecules in HDLp-A do not exchange with free apoLp-111, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-I11 in HDLp-A.
Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in ... more Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d-1.109 g/ml) and by a low density lipophorin during flight (LDLp, d-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 t o reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d-1.118 g/ml) or diacylglycerol (d-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-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-I11 associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-111. 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.-van Heusden, M. C., D. J. van der Horst, J. K. Kawooya, and J. H. Law. In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.
Adult M~nduca sexto high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-... more Adult M~nduca sexto high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I,-11, and-111) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-111. 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 A, 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-111. The particles retained apoLp-I and-11 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: I) all phospholipid and apoLp-I11 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.
Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to fli... more Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to flight muscle, a process mediated by the adipokinetic hormone. An adult specific high density lipophorin (lipoprotein, HDLp-A, Mr = 7.68 X 10(5)) accepts diacylglycerol from the fat body, increasing in size and decreasing in density, to give a low density lipophorin (lipoprotein, LDLp, Mr = 1.56 X 10(6)). During this process, several molecules of the small apolipoprotein, apolipophorin III (apoLp-III), are added to the two molecules originally present in HDLp-A. A study of the time course of adipokinetic hormone-induced loading of diacylglycerol onto HDLp-A, using the analytical ultracentrifuge and gel filtration, suggests that a lipoprotein of density intermediate between HDLp-A and LDLp was formed transiently. Analysis of lipoproteins separated by density gradient ultracentrifugation in the course of the loading process indicates that apoLp-III is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-III, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-III adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-III subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-III showed that the two apoLp-III molecules in HDLp-A do not exchange with free apoLp-III, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-III in HDLp-A.
Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total... more Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total egg lipid is derived from de novo synthesis by the follicles. The remaining egg lipid originates in the fat body and is transported to the ovary by lipoproteins. Vitellogenin, the major egg yolk lipoprotein, accounts for 6% of the total egg lipid. The remaining 95% lipid is attributable to the hemolymph lipophorins, adult high density lipophorin (HDLp-A) and low density lipophorin (LDLp). When HDLp-A that is dual labeled with 'H in the diacylglycerol fraction and "S in the protein moiety is incubated with follicles in vitro, the ratio of SH:S6S in the incubation medium does not vary and is similar to the ratio of the labels that are associated with the follicles. In an accompanying paper (Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) J. Biol. Chem. 263,8740-8747), we show that HDLp-A is sequestered by the follicles without subsequent hydrolysis of its apoproteins. These results, together with those presented in this paper, support our conclusion that HDLp-A is not recycled back into the hemolymph after it is internalized by the follicles and, therefore, does not function as a reusable lipid shuttle between the fat body and the ovary. When follicles are incubated with dual labeled LDLp, the diacylglycerol component of the particle is internalized by the follicles without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by which lipid is delivered to the ovary. Vitellogenin, the precursor of the insect egg yolk protein, and lipophorin, the major insect hemolymph lipid-carrying protein, accumulate in large amounts in insect eggs during oogenesis, These proteins are synthesized in the fat body and are secreted into the hemolymph from which they are specifically internalized by the maturing follicles (
The egg of Manduca sexta contains a very high density lipophorin (VHDLp-E; M, = 4.14 X lo6, p = 1... more The egg of Manduca sexta contains a very high density lipophorin (VHDLp-E; M, = 4.14 X lo6, p = 1.238 g/ml) that is derived from the high density lipophorin (HDLp-A; M, = 7.63 X lo6, p = 1.076 g/ml) of the hemolymph. The selective uptake of HDLp-A into the egg and its subsequent conversion to VHDLp-E was studied both in vivo and in vitro. Upon entering the egg, an estimated 530 mol of lipid were stripped from each mol of HDLp-A, and 68% of the diacylglycerol fraction was converted to triacylglycerol. In addition, the two molecules of the low molecular weight apolipoprotein, apolipophorin-111, of HDLp-A were dissociated from the lipophorin particle. The VHDLp-E thus formed consisted of 80% protein and 20% lipid, 75% of which was phospholipid. HDLp-A labeled in vivo with [SsS]methionine in its apoprotein moiety was injected into females at the onset of egg development, and its incorporation in a series of follicles at different stages of growth was measured. There was increased accumulation of ["SIHDLp-A in the follicles as they matured. The apoproteins of [S6S]HDLp-A were not hydrolyzed when the particle was internalized by the follicle. In the accompanying paper we have presented the evidence that the apoproteins of HDLp-A are retained in the follicles (Kawooya,
Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular ... more Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purifi...
Publisher Summary This chapter discusses the structure and function of major groups of hemolymph ... more Publisher Summary This chapter discusses the structure and function of major groups of hemolymph proteins that are common to all insects, storage proteins, lipoproteins, vitellogenins, and inducible antibacterial proteins. It also discusses some proteins and peptides that are present in smaller amounts, sometimes occurring only in a few insect species. With the development of microsequencing techniques that can provide information about the sequence of amino acids at the N-terminal end of an intact protein on a sample of less than 20 picomoles, it has become feasible to use one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) a the protein purification method of choice. In many cases, proteins can be transferred directly from gels to derivatized paper or other media and spots cut from the medium can be inserted directly into the sequencer. If necessary, proteins on the medium can be cleaved to peptides, which can be separated by high-pressure liquid chromatography (HPLC) or by PAGE.
The hemolymph of adult Munducu aextu (tobacco hornworm) contains a 17,000-dalton protein that can... more The hemolymph of adult Munducu aextu (tobacco hornworm) contains a 17,000-dalton protein that can associate reversibly with the insect lipoprotein lipophorin. The protein is abundant in the hemolymph of the adult, but is found in larval hemolymph in only small amounts, and does not associate with larval lipophorin. On the basis of its association with adult lipophorin, we have designated the protein apolipophorin 111. Apolipophorin I11 was dissociated from adult lipophorin by guanidinium chloride treatment and isolated by gel permeation and ion exchange chromatography. The unassociated apolipophorin I11 was also purified from lipophorin-free hemolymph by gel permeation, ion exchange, and lectin chromatography. Both preparations have identical isoelectric points and amino acid composition as well as the following properties. Apolipophorin I11 is a non-glycosylated polypeptide lacking cysteine and tryptophan. The 17,000-dalton polypeptide dimerizes in solution to a protein of Mr *This study was supported by Grant 29238 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Two cercaria emitted from the snail Melanoides tuberculata are illustrated, one of which encysts ... more Two cercaria emitted from the snail Melanoides tuberculata are illustrated, one of which encysts on the gills of the fish Tilapia zilli. The parasite is shown to have a marked preference for the anterior gills and especially the middle part of each gill. Comparisons between the respiration and ventilation rates of experimentally infected and uninfected fish were made at two levels of activity. At the slower flow rate the parasitized fish have significantly higher oxygen consumptions than do the uninfected controls.
larval stage of the cotton leaf-worm Spodoptera littoralis (Boisd.) (Noctuidae, Lepidoptera) in A... more larval stage of the cotton leaf-worm Spodoptera littoralis (Boisd.) (Noctuidae, Lepidoptera) in Alexandria region. 2. ang. Ent. 74, 332-336. HOSNEY, M. M.; KOTBY, F. A., 1960: Host plants favoured by cotton leafworm moth, Prodenia litura F., for egg-laying, and their value as egg-mass trap crop. Bull. SOC. Ent. Egypte 44, 223-234.
The effect of different factors, such as stage of development, age, sex and pregnancy, on zymogra... more The effect of different factors, such as stage of development, age, sex and pregnancy, on zymograms of the tsetse fly, was studied for eight different enzyme systems. The study provided information with respect to the collection of material in the field and subsequent storage and transport to the laboratory.In a laboratory strain of G. morsitans, genetic polymorphism was demonstrated for five different gene loci, involved in the control of leucine‐amino peptidase, malic enzyme, and alkaline phosphatase. Several allozymes were found to occur at the lap3 locus and at the me‐locus.RésuméVARIATION GENETIQUE EN DIVERS SYSTEMES ENZYMATIQUES DANS LA MOUCHE TSETSE GLOSSINA MORSITANSL'effet de différents facteurs comme le stade de développement, l'âge, le sexe et l'état gravide sur les zymogrammes de la mouche Tsétsé a été étudié pour divers systèmes enzymatiques. Cette étude donne des informations en relations avec le matériel récolté sur le terrain et aussi après le stockage et...
The catalytic domain of human tumor necrosis factor- (TNF-) converting enzyme (TACE) was expresse... more The catalytic domain of human tumor necrosis factor- (TNF-) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF- to generate the mature TNF- in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF--specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative effica...
sity gradient ultracentrifugation in the course of the loading process indicates that apoLp-I11 i... more sity gradient ultracentrifugation in the course of the loading process indicates that apoLp-I11 is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-111, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-I11 adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-111 subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-I11 showed that the two apoLp-I11 molecules in HDLp-A do not exchange with free apoLp-111, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-I11 in HDLp-A.
Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in ... more Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d-1.109 g/ml) and by a low density lipophorin during flight (LDLp, d-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 t o reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d-1.118 g/ml) or diacylglycerol (d-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-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-I11 associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-111. 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.-van Heusden, M. C., D. J. van der Horst, J. K. Kawooya, and J. H. Law. In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.
Adult M~nduca sexto high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-... more Adult M~nduca sexto high density lipophorin (HDLp-A) is composed of three apolipoproteins (apoLp-I,-11, and-111) and 52% lipid. The flight-specific low density lipophorin (LDLp) contains 62% lipid and is associated with several additional molecules of apoLp-111. 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 A, 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-111. The particles retained apoLp-I and-11 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: I) all phospholipid and apoLp-I11 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.
Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to fli... more Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to flight muscle, a process mediated by the adipokinetic hormone. An adult specific high density lipophorin (lipoprotein, HDLp-A, Mr = 7.68 X 10(5)) accepts diacylglycerol from the fat body, increasing in size and decreasing in density, to give a low density lipophorin (lipoprotein, LDLp, Mr = 1.56 X 10(6)). During this process, several molecules of the small apolipoprotein, apolipophorin III (apoLp-III), are added to the two molecules originally present in HDLp-A. A study of the time course of adipokinetic hormone-induced loading of diacylglycerol onto HDLp-A, using the analytical ultracentrifuge and gel filtration, suggests that a lipoprotein of density intermediate between HDLp-A and LDLp was formed transiently. Analysis of lipoproteins separated by density gradient ultracentrifugation in the course of the loading process indicates that apoLp-III is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-III, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-III adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-III subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-III showed that the two apoLp-III molecules in HDLp-A do not exchange with free apoLp-III, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-III in HDLp-A.
Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total... more Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total egg lipid is derived from de novo synthesis by the follicles. The remaining egg lipid originates in the fat body and is transported to the ovary by lipoproteins. Vitellogenin, the major egg yolk lipoprotein, accounts for 6% of the total egg lipid. The remaining 95% lipid is attributable to the hemolymph lipophorins, adult high density lipophorin (HDLp-A) and low density lipophorin (LDLp). When HDLp-A that is dual labeled with 'H in the diacylglycerol fraction and "S in the protein moiety is incubated with follicles in vitro, the ratio of SH:S6S in the incubation medium does not vary and is similar to the ratio of the labels that are associated with the follicles. In an accompanying paper (Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) J. Biol. Chem. 263,8740-8747), we show that HDLp-A is sequestered by the follicles without subsequent hydrolysis of its apoproteins. These results, together with those presented in this paper, support our conclusion that HDLp-A is not recycled back into the hemolymph after it is internalized by the follicles and, therefore, does not function as a reusable lipid shuttle between the fat body and the ovary. When follicles are incubated with dual labeled LDLp, the diacylglycerol component of the particle is internalized by the follicles without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by which lipid is delivered to the ovary. Vitellogenin, the precursor of the insect egg yolk protein, and lipophorin, the major insect hemolymph lipid-carrying protein, accumulate in large amounts in insect eggs during oogenesis, These proteins are synthesized in the fat body and are secreted into the hemolymph from which they are specifically internalized by the maturing follicles (
The egg of Manduca sexta contains a very high density lipophorin (VHDLp-E; M, = 4.14 X lo6, p = 1... more The egg of Manduca sexta contains a very high density lipophorin (VHDLp-E; M, = 4.14 X lo6, p = 1.238 g/ml) that is derived from the high density lipophorin (HDLp-A; M, = 7.63 X lo6, p = 1.076 g/ml) of the hemolymph. The selective uptake of HDLp-A into the egg and its subsequent conversion to VHDLp-E was studied both in vivo and in vitro. Upon entering the egg, an estimated 530 mol of lipid were stripped from each mol of HDLp-A, and 68% of the diacylglycerol fraction was converted to triacylglycerol. In addition, the two molecules of the low molecular weight apolipoprotein, apolipophorin-111, of HDLp-A were dissociated from the lipophorin particle. The VHDLp-E thus formed consisted of 80% protein and 20% lipid, 75% of which was phospholipid. HDLp-A labeled in vivo with [SsS]methionine in its apoprotein moiety was injected into females at the onset of egg development, and its incorporation in a series of follicles at different stages of growth was measured. There was increased accumulation of ["SIHDLp-A in the follicles as they matured. The apoproteins of [S6S]HDLp-A were not hydrolyzed when the particle was internalized by the follicle. In the accompanying paper we have presented the evidence that the apoproteins of HDLp-A are retained in the follicles (Kawooya,
Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular ... more Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purifi...
Publisher Summary This chapter discusses the structure and function of major groups of hemolymph ... more Publisher Summary This chapter discusses the structure and function of major groups of hemolymph proteins that are common to all insects, storage proteins, lipoproteins, vitellogenins, and inducible antibacterial proteins. It also discusses some proteins and peptides that are present in smaller amounts, sometimes occurring only in a few insect species. With the development of microsequencing techniques that can provide information about the sequence of amino acids at the N-terminal end of an intact protein on a sample of less than 20 picomoles, it has become feasible to use one- or two-dimensional polyacrylamide gel electrophoresis (PAGE) a the protein purification method of choice. In many cases, proteins can be transferred directly from gels to derivatized paper or other media and spots cut from the medium can be inserted directly into the sequencer. If necessary, proteins on the medium can be cleaved to peptides, which can be separated by high-pressure liquid chromatography (HPLC) or by PAGE.
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Papers by John Kawooya