Hans Emmerich

Purification and properties of oocyte vitellin from the migratory locust

Summary1.A simple and rapid method is described for the isolation and purification of oocyte vitellin ofLocusta migratoria. The isolated protein has been shown to be homogenous by polyacrylamide gel electrophoresis, isoelectric focusing, sedimentation analysis and in the Ouchterlony test.2.The yolk protein stains with Sudan black and lipid crimson, it reacts with the PAS-reagent and is thus a lipo-glycoprotein. Its isoelectric point is at pH 6.9. At neutral pH the protein is poorly soluble in solutions of low ionic strength, but is easily soluble at alkaline pH. At neutral or acidic pH the yolk protein tends to aggregate to a dimer and a trimer.3.The amino acid composition shows a high content of aspartic and glutamic acid or their amides and a low percentage of sulphur containing amino acids. As N-terminal amino acids alanine and aspartic acid are found.4.The yolk protein consists of several non-identical subunits. In polyacrylamide gel electrophoresis with sodium dodecyl sulphate subunits of 55,000, 65,000, 110,000, 120,000 and 130,000 Daltons are found. The molecular weight was determined to 530,000±30,000 Daltons, the sedimentation coefficient ass20,w=16.3±0.02 (corrected). The frictional ratio isf/f0=1.105, the molar extinction coefficient at 280 nm is 4.2×105 (=0.91 per mg protein). All subunits stain as glycoproteins; the total sugar content was determined as 11%.

Changes in the titer of vitellogenin and of diglyceride carrier lipoprotein in the blood of adult Locusta migratoria

Abstract Vitellogenin (VG) and the diglyceride carrying lipoprotein (DGCL) are the predominant lipoproteins in the haemolymph of adult female locusts. Antibodies against these two proteins have been prepared which do not reveal any cross reactivity. The titer of these haemolymph proteins was followed during two ovipository cycles by quantitative rocket electrophoresis. The titer of DGCL reaches a constant level of 12 mg/ml three days after adult emergence, while the vitellogenin titer undergoes characteristic changes correlated with the growth of the terminal oocytes. Until day 8 vitellogenin is virtually absent in the blood, it then quickly rises to a maximum of 20 mg/ml at day 11, corresponding to an oocyte length of 3.5 mm. Within a further 6 hr the vitellogenin concentration drops to 4 mg/ml. With the onset of the next ovipository cycle these changes in VG concentration occur again.

Differences in Hydrolysis and Binding of Homologous Juvenile Hormones in Locusta migratoria Hemolymph

Abstract Male Locusta hemolymph contains JH-esterase(s) and a JH-carrier protein with high affinity and low capacity that both interact enantioselectively with juvenile hormones. Exposure of race-mic JH-I and JH-III results in preferential hydrolysis of the naturally configurated enantiomer of JH-I but the unnaturally configurated enantiomer of JH-III. The JH-carrier protein has a remark able specificity for the natural enantiomer of JH-III but discriminates only weakly between enantiomers of JH-I. This observation indicates a protective function of the JH-specific carrier protein for JH-III but not for JH-I. A third major protein, the diglyceride carrier lipoprotein (DGCL), shows no stereoselectivity at all.

Immunohistochemical localisation of ecdysteroids in the follicular epithelium of locust oocytes.

SummaryFrozen sections of growing terminal follicles of the locust ovary were incubated with an ecdysteroid-specific rabbit antibody and the bound antibody visualised by the use of FITC-labelled goat-anti-rabbit antiserum. A bright fluorescence was seen in the cytoplasm of the follicle cells in terminal follicles with a length between 4.0 and 6.0mm with a maximum intensity at 5.5mm, indicating the presence of ecdysteroids in these cells in this particular developmental stage.

Juvenile hormone metabolism and juvenile hormone esterase titer in hemolymph and peripheral tissues ofDrosophila hydei

SummaryThird larval instar hemolymph of the fruitflyDrosophila hydei did not metabolize juvenile hormone (JH) at all developmental stages. In contrast, prepupal and pupal body fluid showed JH-esterase activity with a maximum at 4 h after puparium formation. In body wall and fat body of all developmental stages investigated, JH-metabolic activity was found. In both tissues JH catabolism was most active in the 120,000g supernatant and pellet. The 800g and 15,000g pellet showed a lower activity. In all subcellular fractions the JH-acid was identified as the predominant metabolite. There is evidence that JH-specific esterases are responsible for ester cleavage in the 120,000g supernatant. During mid and late third larval instar development in both body wall and fat body JH-esterase activity remains relatively constant.

Juvenile hormone binding proteins in the haemolymph of third instar larvae of Drosophila hydei

Abstract Juvenile hormone I (JH I) is bound in vitro by two protein fractions in the haemolymph of mid-third instar larvae of Drosophila hydei. These binding proteins were characterized by gel filtration, density gradient centrifugation, isoelectric focusing, polyacrylamide gel electrophoresis and charcoal adsorption assay. The larger binding protein has a molecular weight of 1.08 × 10 5 , a sedimentation coefficient of approximately 7 S and an isoelectric point at pH 5.1. The equilibrium dissociation constant for JH I is about > 10−5 M. This protein binds JH I after storage for five days at 0 C, but binding capacity is destroyed by heating to 60°C for 10 min. Proteolytic peptides of this binding protein still possess the capacity to bind JH. The smaller JH-binding protein has a molecular weight of 4.4 × 10 4 , a sedimentation coefficient in the range of 3–4 S, an isoelectric point at approximately pH 8.9 and an equilibrium dissociation constant of approximately 1 × 10 −7 M. Its binding capacity is rapidly lost in total haemolymph and after partial purification at 4°C.

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

SummaryInsoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.

Characterization of the acid phosphatase and arylsulphatase activities in a tumorous blood cell line of Drosophila melanogaster

Abstract The lysosomal acid phosphatase (AP) and arylsulphatase (AS) activities in a tumorous blood cell line of Drosophila melanogaster have been characterized. The AP activity measured with para -nitrophenylphosphate as substrate had a pH optimum of pH 4. The K m for this substrate was 0.1 mM. The activity was inhibited by l -tartrate, fluoride ions, DFP and anions such as phosphate and molybdate, but not by sulphydryl reagents nor by alkaline phosphatase inhibitors. The activity was adsorbed onto DEAE-Sephadex at pH 8 and eluted as one peak, when a NaCl gradient was applied to the column. It was not possible to separate the activity into several components by disc-gel electrophoresis or isoelectric focusing (pI of AP: 6.2). The mobility during disc-gel electrophoresis was not altered by prior incubation with bacterial neuraminidase. The AS was shown to hydrolyse the artificial substrates para -nitrophenylsulphate, para -nitrocatecholsulphate and acetylphenyl sulphate with pH optima of 6.8–7.2. The activity was detrimentally affected by various inorganic anions and could be totally inhibited by low concentrations of AgNO 3 (10 −4 M). The AS bound to the anion exchanger more strongly than the AP, but in common with the AP, only one peak of activity could be detected by this and other separation techniques. The pI of the AS was 4.6. The AP and AS have similar apparent molecular weights (60–65,000) and sedimentation coefficients (6.6 and 5.8S, respectively).

Derivatives of the insect moulting hormone for affinity chromatography, and their biological activities.

Several derivatives of the arthropod moulting hormone have been synthesized which were coupled to AH Sepharose 4B yielding about 2 mumole ligand per g wet gel. As an indication of the suitability of the ligands for biological work the puff inducing capacity of their methyl esters was tested. The methyl ester of inokosterone-C-26-carboxylic acid possesses the highest biological activity; lower activities were obtained with the esters of ecdysterone-C-6-CM-oxime and ecdysterone hemisuccinates. Therefore, inokosterone-C-26-carbocylic acid should be a useful ligand for affinity chromatography of ecdysone recptors from insect tissues.

Immunofluorescent distribution of postribosomal particles during oogenesis and early development of an insect (Dysdercus intermedius, Heteroptera, Pyrrhoc.)

SummaryDuring previtellogenesis, the oocytes of the telotrophic meroistic ovary ofDysdercus are provided with ribosomes and ribonucleoprotein (RNP) particles by the nurse cells. At the end of vitellogenesis, the oocyte itself becomes active as shown by autoradiography. The proteins synthesized by the oocyte are stored in cytoplasmic postribosomal particles which are preformed by the tropharium. The proteins of these particles were separated by SDS polyacrylamide gels and their endogenous oocyte proteins revealed by fluorography. The synthesis, transport, and storage of the postribosomal particles are demonstrated by indirect immunofluorescence. The young oocytes of previtellogenic follicles show a diffuse distribution of these particles. In late vitellogenesis, fluorescence becomes more and more concentrated in spots throughout a distinct region in the middle part of the oocyte. Thus, in freshly laid eggs, the periplasm is free of fluorescence. During migration of the cleavage nuclei the postribosomal particles were shifted into the cortex. Fluorescence is then most intense in the periplasmic region. During blastoderm formation, however, fluorescence decreases.