Evidence for vitellin maturation within oocytes of Perinereis cultrifera (Polychaete annelid)

• 1.1. The relationship between the five native forms of vitellin (V₁-V₅) previously identified in Perinereis cultrifera oocytes was studied by following the distribution of [³H]leucine between these vitellin forms after in vivo injection of the amino acid in young and submature females.
• 2.2. By native polyacrylamide gel electrophoresis and fluorography, the highest molecular weight form of vitellin (V₁, 530,000) was first (one day incubation period) detected as being radioactive while for long incubation periods (3 to 14 days) a progressive shift in labelling was observed from V₁ into the smaller forms of vitellin (V₂, 500,000; V₃, 470,000; V₄, 430,000 and V₅, 390,000).
• 3.3. During the increasing incubation periods, a progressive shift in labelling from a single high molecular weight polypeptide (P₁, 176,000) into lower molecular weight fragments characteristic of the mature vitellin form V₅ was found when the vitellin fractions obtained by immunoprecipitation were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and fluorography.
• 4.4. These results confirm the hypothesis of a precursor-product relationship between V₁ and V₅ via three intermediate forms (V₂ to V₄) and strongly suggest that the conversion process within the oocyte involves progressive proteolytic cleavages of a single precursor component, or polypeptide subunit of V₁, into those that make up V₅, the mature form of vitellin.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Three digestive tract endocrine peptides and the circannual cycle of body weight in Spermophilus lateralis

• 1.1. Thirty-one male golden-mantled ground squirrels were divided into four physiological groups: low wt summer, medium wt summer, high wt summer and hibernation period. A second group of 10 females was divided into two groups: hibernation period at low T_b and hibernation period during a periodic arousal.
• 2.2. Blood serum, pancreas and antral stomach region were collected from each animal.
• 3.3. The serum was analysed by radioimmunoassay for pancreatic polypeptide immunoreactivity, the pancreas for pancreatic polypeptide and somatostatin immunoreactivity and the antral region of the stomach for gastrin immunoreactivity.
• 4.4. Significant between-stage differences (P < 0.05) were found in serum pancreatic polypeptide concentration and in pancreatic somatostatin content.

     

Immunocytochemical localisation of vitellogenic and non-vitellogenic yolk proteins in the ovary of leptinotarsa decemlineata

• 1.1. Two vitellogenins and chromoprotein 2 are selectively accumulated by the oocyte and cannot be detected either in follicle cells or in the germarium.
• 2.2. At the start of their accumulation in terminal oocytes they are asymmetrically distributed.
• 3.3. Endocytosis of vitellogenin 1 starts somewhat later than the uptake of vitellogenin 2 and chromoprotein 2.
• 4.4. In follicle cells of young follicles, a protein (DLP), immunologically related to diapause protein 1, is highly concentrated.
• 5.5. During vitellogenesis DLP is sequestered by the oocytes.
• 6.6. The protein rich globules in terminal oocytes contain the vitellins as well as chromoprotein 2 and DLP.

     

Synthesis and maturation of the major yolk protein by the ovaries of the firebrat,Thermobia domestica (Insecta,thysanura)

• 1.1. Ovaries of Therobia domestica, dissected from inseminated females and incubated with tritiated amino acids, synthesize labeled proteins, the major fraction of which is indistinguishable from the major vitellogenin secreted by the fat body, when considering the electrophoretic mobility, the polypeptide composition and the immunoreactivity.
• 2.2. Peptide mapping, using two different proteases, shows a striking structural similarity between the proteins of both origins and reveals interrelationships between their subunits.
• 3.3. The ovary synthesizes the 210–212 kD precursors of the major vitellogenin, as does the fat body, and processes them intensively into smaller subunits (176–182, 57 and 46 kD). The follicle cells are tentatively nominated for both roles.
• 4.4. The quantitative contribution of the two ovaries to the vitellogenin pool was found to be much higher than that of the fat body.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.

     

Variation in plasma constituents during the natural vitellogenic cycle of tuatara,Sphenodon Punctatus

• 1.1. The vitellogenic cycle of female tuatara was investigated by monitoring plasma levels of vitellogenin, calcium, total protein, inorganic phosphate (P₁) and cholesterol.
• 2.2. Vitellogenin was not detected in females in the non-reproductive condition, but was found perenially in plasma of reproducing females during vitellogenesis, which normally lasts about 3 years out of the 4 year ovarian cycle.
• 3.3. No large year-to-year variations were found in the plasma constituents measured and there was no correlation between the oestradiol peak at mating and plasma levels of vitellogenin.
• 4.4. The results provide further evidence that tuatara have an extraordinary prolonged and gradual vitellogenic cycle spanning several years for a single clutch of eggs. This type of reproductive cycle is unique among reptiles.

     

Identification and partial characterization of plasma membrane polypeptides of Crithidia guilhermei,crithidia deanei and Crithidia oncopelti

• 1.1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE).
• 2.2. Significant differences were noted in the proteins present in the DRP when the three species were compared.
• 3.3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti.
• 4.4. One polypeptide with mol. wt 50,000 and p1 4.9 was identified in the DRP of C. deanei.
• 5.5. A broad band with mol. wt 68,000–140,000 and pI 4.7–5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed.
• 6.6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti.
• 7.7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Comparative vitellin pattern and cladogenesis in Bacillus (insecta: Phasmatodea)

• 1.1. Under denaturing conditions (SDS-PAGE) the two natural vitellins of Bacillus taxa released five different polypeptides (A₁, A₂, A₃, B₁, B₂).
• 2.2. A₂ and B₂ bands from the two bisexual species (B. rossius and B. grandii) were found to differ; furthermore a non-vitellin yolk protein characterizes the subsepecies B.g. benazzii.
• 3.3. From gels and their densitometric scanning profiles it is clear that parental polypeptides are expressed in the thelytokous parthenogenetic hybrids (B. whitei, B. lynceorum) and in the hybridogenetic B. rossius-grandii benazzii.
• 4.4. A comparative approach of vitellin patterns appears fully adequate for tracing phylogenetic relationships and recognizing cladogenetic events.

     

Partial characterization and subunit analysis of major phosphoproteins of egg yolk in the fish,Oryzias latipes

• 1.1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka (Oryzias latipes) eggs were performed.
• 2.2. PP1 (M_r ≈ 210,000) and PP2 (M_r ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively.
• 3.3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ.32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively.
• 4.4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Chromatin proteins of rat preputial-gland: Acute changes in response to estrogen

• 1.1. The effects of estradiol-17β (E₂β) at 2 or 15min in vivo on chromatin proteins of rat preputial-gland were analyzed by a battery of electrophoretic methods.
• 2.2. Among histones, E₂β/control ratios for major bands of H1 decreased substantially between 2 and 15 min. In contrast, ratios of H4 increased (P < 0.01), whereas, except for losses by 2 min m a H2B-like component and in H3.1, other core histones were unchanged.
• 3.3. Among 0.35 M NaCl-soluble proteins, components at 34K-mol. wt and < 21K-mol wt were increased after 2 min of E₂β. The bulk of the hormone-responsive low-molecular weight proteins was basic in charge.
• 4.4. Electrophoretic correlates of 6 basic lysosomal proteins corresponded to those of low-molecular weight salt-soluble chromatin proteins.
• 5.5. Selective proteolysis initiated in vivo by E₂β depleted some tightly-bound nonhistone proteins.

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

Fate of ingested leucine and glucose in the sea star,Asterias rubens,during its annual reproductive cycle

• 1.1. The distribution of radiolabel from L-leucine [¹⁴C-UL] and D-glucose [¹⁴C-UL] was measured in the sea star Asterias rubens at 1, 6 and 24 hr after oral administration.
• 2.2. Incorporation of the label from both compounds was observed in pyloric caeca, coelomocytes and ovaries even after an incubation time of 1 hr.
• 3.3. Highest incorporation from both precursors was found in proteins, while substantial radioactivity was present in the amino acids, organic acids and neutral components. Lipids were hardly labelled from leucine and only slightly from glucose.
• 4.4. Radioactivity in proteins and lipids increased with increasing incubation time. No significant differences were found in the distribution patterns of radiolabel during the reproductive cycle.
• 5.5. The data obtained are discussed in terms of current knowledge on the translocation of nutrients in echinoderms.

     

Monoclonal antibodies to antigen binding protein of annelids (Lumbricus terrestris)

• 1.1. Monoclonal antibodies (mAb) to antigen binding protein (ABP) of the earthworm Lumbricus terrestris have been prepared.
• 2.2. The specificity of mAb for a determinant located outside the antigen binding site was determined and verified in inhibition experiments.
• 3.3. The mAb were used for isolation of a 56 kDa ABP by an immunoprecipitation technique.
• 4.4. The binding of mAb to coelomocytes has demonstrated the existence of two cell populations, one with low and the other with high densities of ABP molecules on the cell membranes.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.