Polypeptide composition and biosynthesis of the yolk proteins in the firebrat,Thermobia domestica (insecta,thysanura)

Ion-exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (M_r 166,000–212,000) and small (around M_r 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse-chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at M_r 210,000 and 212,000, is rapidly processed to M_r 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.     

An IS4-encoded protein is synthesized in minicells

Summary A protein of M_r 47,000 is synthesized in Escherichia coli minicells, when these harbor a multicopy plasmid carrying IS4 in either orientation and between different flanking sequences. The protein corresponds to the sequence predicted from the known DNA sequence of IS4, as shown by partial N-terminal radiolabel protein sequence analysis. Its apparent molecular weight, however, as determined from its electrophoretic mobility in SDS polyacrylamide gels, is smaller than predicted. When compared with other plasmid-encoded proteins, the IS4-encoded protein is synthesized in minicells in small amounts. Its synthesis has not been detected in a DNA-dependent cell-free system.     

Purification and Characterization of an endo-Polygalacturonase from the Gut of West Indies Sugarcane Rootstalk Borer Weevil (Diaprepes abbreviatus L.) Larvae

An endo-polygalacturonase (PG) (EC:3.2.1.15) with a pI of 9.4 and an M_r of 44,500 was purified to electrophoretic homogeneity from the gut of West Indies sugarcane rootstalk borer weevil (Diaprepes abbreviatus L.) larvae. Hydrolytic activity was maximal in 150 mM sodium acetate, pH 5.5, at 30°C. Kinetic determinations yielded an apparent K_m of 3.68 mg polygalacturonic acid (PGA)/ml and a V_max of 283 μmol galacturonic acid/min/mg protein for PGA. Enzymatic activity was inhibited by a polygalacturonase inhibitor protein from “Hamlin” orange flavedo. The purified protein does not appear to be glycosylated, and its N-terminal sequence showed no homology to any PG protein sequences in data banks.     

Characterization and comparison of arcelin seed protein variants from common bean

Four variants of arcelin, an insecticidal seed storage protein of bean, Phaseolus vulgaris L., were investigated. Each variant (arcelin-1, -2, -3, and -4) was purified, and solubilities and M_rs were determined. For arcelins-1, -2, and -4, the isoelectric points, hemagglutinating activities, immunological cross-reactivities, and N-terminal amino acid sequences were determined. On the basis of native and denatured M_rs, the variants were classified as being composed of dimer protein (arcelin-2), tetramer protein (arcelins-3 and -4), or both dimer and tetramer proteins (arcelin-1). Although the dimer proteins (arcelins-1d and -2) could be distinguished by M_rs and isoelectric points, they were identical for their first 37 N-terminal amino acids and had similar immunological cross-reactions, and bean lines containing these variants had a DNA restriction fragment in common. The tetramer proteins arcelin-1t and arcelin-4 also could be distinguished from each other based on M_rs and isoelectric points; however, they had similar immunological cross-reactions and they were 77 to 93% identical for N-terminal amino acid composition. The similarities among arcelin variants, phytohemagglutinin, and a bean α-amylase inhibitor suggest that they are all encoded by related members of a lectin gene family.     

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus

Summary The amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 M_r polypeptide of CLV-1 (3) and the 40 285 M_r polypeptide of TGMV-A (4) show extensive homologies with the 17 768 M_r and 31 388 M_r polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.     

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin , was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M _r 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin . It is concluded that conglutin is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.     

Diverse dextranase genes from <Emphasis Type="Italic">Paenibacillus</Emphasis> species

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M_r of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M_r of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Characterization of the Major Protein Component from Aleurone Cells of Barley (Hordeum vulgare L.)

Comparison of the SDS-PAGE patterns of salt-soluble proteinsfrom aleurone protoplasts, enriched aleurone layers preparedby pearling, and isolated embryos of mature barley showed threegroups of bands that reacted with antiserum raised against the7S globulins of oat embryos. These had M_rs of about 50 000,40 000 and 25 000. The enriched aleurones also contained a thirdgroup of immunoreactive bands (M_r about 70 000), which did notco-purify when the proteins were fractionated by ammonium sulphateprecipitation, ion exchange chromatography and immunoaffinitychromatography. The purified protein gave a single sharp peakon RP-HPLC, and contained a fourth minor group of subunits ofM_r about 20 000, in addition to those of M_rs about 50 000, 40000 and 25 000. The holoprotein and the ‘major groups’ of subunitsall had similar major N-terminal amino acid sequences whichwere related to the N-terminal amino acid sequences of pea andbean vicilins, and to sequences in the putative N-terminal regionsof the mature 7S ¹-globulins of cottonseed, confirming the homologywith these groups of proteins. Key words: Barley, seeds, 7S globulin, vicilin, ¹-globulin, amino acid sequence, protein homology     

Protein processing as a regulatory mechanism in the synthesis of the photosynthetic antenna in Chloroflexus

Chloroflexus aurantiacus can be induced to shift from respiratory to photosynthetic energy production by introducing light and/or lowering the oxygen concentration of a culture. After induction, cells synthesize bacteriochlorophyll and proteins for the formation of a functional photosynthetic apparatus. Bacteriochlorophyll is detectable within 2 h after induction. Chlorosome polypeptides are detected after 8–12 h. Two proteins, M_r 60,000 and M_r 47,000, are present in both induced and noninduced cells and react specifically with antibodies against chlorosome polypeptides. Immunological data suggest that these proteins (M_r 60,000 and 47,000) are polyproteins which are transcribed and translated in the dark. When cells are exposed to light or low oxygen tension these proteins are processed into functional polypeptides required in the assembly of the chlorosome. The reaction center polypeptide (M_r 26,000) appears to be part of a separate genetic control system.Dedicated to Prof. G. Drews on occasion of his 60th birthday     

Isolation and partial characterization of nigrin b,a non-toxic novel type 2 ribosome-inactivating protein from the bark ofSambucus nigra L.

The bark ofSambucus nigra L. contains a non-toxic novel type 2 ribosome-inactivating protein that we named nigrin b.In vitro, nigrin b strongly inhibited mammalian protein synthesis but did not affect plant nor bacterial protein synthesis. The protein (M _r 58 000) contains two subunits, A (M _r 26 000) and B (M _r 32 000); linked by disulphide bridge(s). Nigrin b was found to be an rRNA N-glycosidase of the rRNA of intact mammalian ribosomes and shares a very good N-terminal amino-acid sequence homology with the anti-HIV-1 proteins TAP 29 and trichosanthin.     

Preparation, Properties, and Composition of Nereis vitellin, the Yolk Protein of the Annelid, Nereis virens

In the literature, oogenesis in nereid annelids is considered to be a model system because, unlike other system studied, nereid oocytes are thought to synthesize the bulk of their yolk protein themselves. As the first step to test the validity of this hypothesis, nereid yolk protein was characterized biochemically. Vitellin, the main fraction of the soluble yolk proteins, was prepared from Nereis virens oocytes. Preparation, purification, and some physical characteristics of this green-colored protein Nereis vitellin are described. The molecular weight was determined by gel chromatography as 420,000 daltons. With regard to the amino acid composition, Nereis vitellin was found to resemble both insect vitellins and an average protein, as defined by other authors. Methionine and cysteine were found in traces only. By staining procedures, Nereis vitellin was characterized as lipoglycoprotein. Nereis vitellin was also prepared from the coelomic fluid of gravid females of Nereis virens .     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Postendocytic vitellin processing in ovarian follicles of the stick insect Carausius morosus (Br.)

Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A₁, A₂, and A₃) and 2 (B₁ and B₂) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV follicles, Vit A resolved into 4 distinct vitellin polypeptides (A₀, A₁, A₂ and A₃). Using a panel of monoclonal antibodies, polypeptide A₀ proved to be immunologically related to polypeptide A₂. In follicles about to begin choriongenesis, polypeptide A₃ was gradually replaced by a lower M_r polypeptide. Over the same time period, polypeptide B₁ changed in charge, but not in M_r. To confirm the existence of a polypeptide processing in C. morosus, ovarian follicles of different developmental stages were exposed in vivo to [³⁵S]-methionine from 6 to 72 h. Data showed that A₀ and B₁ were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B₂ and A₃ were also labelled to some extent. With progressively longer exposures, polypeptides A₁ and A₂ also became labelled. In vivo exposure to [³H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of ovarian follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.     

Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai

Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai.     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.