Targeting of glutamine synthetase to the mitochondria of transgenic tabacco

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia -F₁ ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the -F₁ ATPase has successfully targeted the GS polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.     

Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain

Summary Certain E. coli K12 strains are able to secrete a plasmid encoded 107 K protein into the culture medium. During exponential growth of the cells this protein represents approximately 1% of total cell protein.The presence of the 107 K polypeptide was demonstrated through the fortuitous use of strain MC4100. This gave a largely protein-free culture supernatant, presumably due to minimal lysis of whole cells. Pulse-labelling experiments showed that the secretion of the 107 K polypeptide reached a maximum during the stationary phase of growth, where it represented substantially more than 1% of total cell protein. The 107 K polypeptide is coded by the haemolytic plasmid pHly167, and appears to be related to a previously reported intracellular precursor form of the -haemolysin (Goebel and Hedgpeth 1982). However, additional extracellular factors appear to be required for -haemolysin activity since several nonhaemolytic mutants still secrete this protein.     

The kell system in Senegal: phenotype and gene frequencies

The Kell gene frequencies, determined in Senegal are as follows: Our data enter the limits already known for black African populations.     

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

Summary The gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5 flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3 end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).     

Barley beta-galactosidase: structure, function, heterogeneity, and gene origin

Barley (Hordeum vulgare) -galactosidase is composed of a large (45 kDa) and a small (33 kDa) polypeptide. N-terminal sequencing of the polypeptides and antibody reactivity data place the barley enzyme and heterodimeric plant -galactosidases from jack bean, maize, and wheat in family 35 of the glycosyl hydrolases. Sequence analysis indicates the existence of a subfamily of genes coding for polypeptide precursors that are cleaved to produce the two subunits in heterodimeric -galactosidases. The heterogeneity of the barley holoenzyme is related, but not restricted, to the N-glycosylation of the small polypeptide. Both polypeptides are essential for the catalytic activity of the enzyme.     

Proteolytic modifications of the B800-860 complex of the photosynthetic bacterium Rhodocyclus tenuis: Structural and spectral effects

The modification effects on the absorption and cirular dichroic (CD) spectra of the isolated B800-860 antenna complex of Rhodocyclus tenuis by a number of proteolytic enzymes were investigated. The chymotrypsin modifications of the B800-860 complex led to an about 40% decrease of the 860-nm band and a blue-shift to 841 nm. The biphasic CD signal related to the B860 BChl disappeared and a new double CD signal with a zero-crossing point at 842 nm appeared. These absorption and CD spectral changes suggested that a B800-841 complex resulted after chymotrypsin digestion. The polypeptide components of the chymotrypsin-modified B800-860 complex were separated by reverse-phase chromatography, and their amino acid sequences determined by protein sequencing and mass spectrometry. Sequence analyses showed that the C-terminal 25 residues of the B800-860- polypeptide and the C-terminal 8 residues of the B800-860- polypeptide were cleaved by chymotrypsin, and the remaining , polypeptide fragments apparently form the structural basis for the newly-formed B800-841 complex. No significant spectral change was observed from exposing the isolated B800-860 complex to trypsin, carboxypeptidase A and the combination of carboxypeptidase A and carboxypeptidase B. Short-term proteinase K incubation of the B800-860 complex of Rc. tenuis led to a preferential decrease of the 860-nm absorbance band and its related CD signals, as compared to the 800-nm absorbance and CD bands, suggesting that the C-terminal portions of the antenna polypeptides are possibly exposed to the exterior of the B800-860 complex micelles. Whereas, long-term proteinase K digestion resulted in the spectral collapse of the B800-860 complex and the release of free BChls. Our proteolysis experiments support the hypothesis that the C-terminal portions of the antenna polypeptides play a key role in the redshift and strong molar extinction of the Q_y band of the B850 BChls.Abbreviations B800-860 light-harvesting complex with the absorption maxima (Q_y) at 800 nm and 860 nm - B800-860- -, polypeptide of the B800-860 complex - CD circular dichroism - Deriphat-160 disodium Nlauryl--iminodipropionate - FT Fourier transform - LH light-harvesting - near-IR near infra-red - OG n-Octyl--glucoside - PTH phenylthiohydantoin - Rb. Rhodobacter - Rc. Rhodocyclus - Rp. Rhodopseudomonas - Rsp Rhodospirillum - DSM Deutsche Sammlung für Mikroorganismen     

Embryonic hemoglobins of different animal species

Summary The existence of embryonic hemoglobins is demonstrated in sheep-, calf and pig embryos. The occurrence and disappearance of these hemoglobins is quantitatively determined by cellulose acetate gel electrophoresis; hemoglobins as well as the globin chains, dissociated in 8 molar urea were quantitated. Sedimentation and diffusion experiments in the analytical ultracentrifuge revealed a S₂₀ of 4.3 and a D₂₀ of 6.6 for the examined hemoglobins. Therefore it is concluded that all hemoglobins occurring at different stages of embryonic and fetal development consist of 4 polypeptide chains with a total molecular weight of 66,000. The subsequent formation of the different polypeptide chains during ontogenesis is shown: At first only -chains are formed as demonstrated by the existence of Hb Gower I, consisting of four identical -chains. Subsequently the -chain appears, which leads to Hb Gower 2 (₂₂). The third polypeptide chain formed during the ontogenesis the -chain results finally in the appearance of HbF.In addition the existence of a HbF pig is demonstrated by the fingerprint technique.     

A new nucleosomal protein in normal liver related to the cytoplasmic polypeptide target of a carcinogen

Summary Normal adult rat liver contains a nucleosomal protein that is related to the principal target polypeptide of a carcinogen in cytoplasm. Normal rat liver was found previously to contain a 14 000-dalton polypeptide that is the principal. cytosolic target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), early during hepatocarcinogenesis. Elevated levels of immunohistochemically detectable target polypeptide in cytoplasm are associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes. A putatively related 17 500-dalton polypeptide was shown to be tightly bound to chromatin of normal liver nuclei. We report here that purified nucleosomes from normal rat liver contain the bound 17 500-dalton protein. Nuclei were digested with micrococcal nuclease, and the resultant nucleosomes were resolved into size classes by density gradient sedimentation. The monomers, dimers, and trimers of nucleosomes possessed bound 17 5019-dalton polypeptide, as determined by SDS gel electrophoresis followed by immunoelectroblot analyses. Alterations in the levels of the two polypeptides were shown previously to occur during liver carcinogenesis by FAA and 3-methyl-4-dimethylaminoazobenzene. The findings support the possibility that the 17 500-dalton polypeptide may function normally in a role related to the replication or expression of the hepatic genome, and may be connected with changes in hepatic genic activity brought about by the carcinogens.Abbreviations FAA N-2-fluorenylacetamide (2-acetylaminofluorene) - 3-Me-DAB 3-methyl-4-dimethylaminoazobenzene - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate     

The β2 subunit of human placenta hexosaminidase (Hex) A and B is a non-random association of the two polypeptides αa and βb

The subunit structures of placental Hex A and B have previously been assigned as _a and _b, respectively. The ₂ subunit is composed of two non-identical polypeptide chains, _a and _b. Purified Hex A and B were fractionated on a chromatofocusing column, and the fractions were reduced and then alkylated with iodo-I-¹⁴C-acetamide. The polypeptide chains were separated by polyacrylamide-gel isoelectric focusing. From the radioactivity measurements of the polypeptides a constant value for ₂ was obtained in all the chromatofocusing fractions, demonstrating a non-random structure of (₂) in each ₂ subunit.     

A software tool for the prediction of Xaa-Pro peptide bond conformations in proteins based on 13C chemical shift statistics

The chemical shift difference ([¹³C] – [¹³C]) is a reference-independent indicator of the Xaa-Pro peptide bond conformation. Based on a statistical analysis of the ¹³C chemical shifts of 1033 prolines from 304 proteins deposited in the BioMagRes database, a software tool was created to predict the probabilities for cis or trans conformations of Xaa-Pro peptide bonds. Using this approach, the conformation at a given Xaa-Pro bond can be identified in a simple NOE-independent way immediately after obtaining its NMR resonance assignments. This will allow subsequent structure calculations to be initiated using the correct polypeptide chain conformation.     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin,synthetic analogs,and a potential intracellular regulatory protein

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H⁺ + K⁺)ATPase. The site of interaction with the (H⁺ + K⁺)ATPase was shown to be the alpha subunit of the (H⁺ + K⁺)ATPase in studies using [¹²⁵I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H⁺ + K⁺)ATPase, and prevented labeling by [¹²⁵I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H⁺ + K⁺)ATPase. These studies help to establish that the (H⁺ + K⁺)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H⁺ + K⁺)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H⁺ + K⁺)ATPase. Using an antibody which recognizes this amphipathic helical (melittin-like) motif, we have demonstrated that the gastric parietal cell contains a 67 kDa melittin-like protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric melittin-like protein appears to overlap with the melittin binding site on the alpha subunit of the (H⁺ + K⁺)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous melittin-like protein on the (H⁺ + K⁺)ATPase to regulated HCl secretion by the parietal cell is presently under investigation. Evidence is presented which demonstrates that melittin binds to other E₁-E₂ ion pumps, raising the possibility that there might exist similar intracellular proteins which interact with other ion pumps.     

Fluorescence metachromasia in polypeptide hormone-producing cells of the APUD series, and its significance in relation to the structure of the precursor protein

Synopsis A fluorescence metachromatic modification of the masked basophilia method is described. It is based on the acridine dye Coriphosphine O. Excitation and emission spectra of green (orthochromatic) and red (metachromatic) fluorescent tissue components are presented.When the method is applied to suitably fixed sections, metachromasia is demonstrable in cells of the polypeptide hormone-secreting APUD series, and in a few other situations.The view that side-chain carboxyl groups are demonstrated by the masked basophilia technique is considered to be accurate but inadequate. It is proposed that the technique, and its fluorescence modification, are influenced more by secondary than by primary protein structure. In particular, it is suggested that the conformation of the protein precursors of polypeptide hormones, in the storage granules of endocrine cells, is predominantly random-coil.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

The effect of “shearing” forces during ultracentrifugation on the membrane-bound polysomes associated with light rough microsomes of MPC-11 cells

When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that [³H]-choline label and A_{2 6 0 nm} absorbing did not coincide in the LR region of the gradient. This was in contrast to the situation when microsomes were isolated from cells disrupted by nitrogen cavitation at 25 mM KCl. The A_{2 6 0 nm} absorbing material that appeared in gradient fractions (1–5) below the position of LR membranes was found to consist of polysomal material. This material gave a richer polysome profile than that released from the LR membranes by addition of detergent. Labelling experiments with [³H]-leucine showed that nascent polypeptides associated with monosomes and polysomes in fraction 1–5 were of shorter length than the corresponding ones in the LR fraction. A mere contamination of LR microsomes by free polysomes appeared most unlikely. The results are consistent with an effect of shearing on the membrane-bound polysomes of the LR microsomes under specific experimental conditions. This effect results in the production of a 5 mRNA fragment (short polypeptide chains) and a 3 mRNA fragment (long polypeptide chains), the former fragment migrating further down the gradient tube free of LR membranes, whilst the latte remained attached to the LR membranes.     

Evolution of antigenic determinants of transferrin and other serum proteins in primates

The antigenic determinants of the transferrins of 23 different primate species have been compared by a radioimmune inhibition of precipitation technique. Highest degrees of cross-reactivity with antisera to human transferrin were found in the transferrin of African apes, followed by that of the Asian apes, the Old World monkeys, the New World monkeys, and finally by the Prosimians. Thus, the evolution of the antigenic determinants on the transferrin molecule is in good agreement with the taxonomic classification of these species. Similar studies were carried out for ₂-macroglobulin and various immunoglobulin chains. Parallel results were obtained except for the heavy chain of M macroglobulin in which gibbon, orangutan, and gorilla probably followed a divergent line of evolution from the other Hominoidea.The work reported in this paper was supported in part by Public Health Service Grants HE 05997 and HE 10486 from the National Heart Institute; the American Cancer Society Grant # T-386A; and a grant from the Life Insurance Medical Research Fund.Recipient, American Cancer Society Postdoctoral Fellowship (PF-407).     

Purification of alpha-hemolysin from an overproducing E. coli strain

Summary The genetic determinant of the -hemolysin encoded by plasmid pHly152 has been cloned in both orientations in plasmid pBR322 giving rise to plasmids pSU157 and pSU158. E. coli strains carrying either of these recombinant Hly plasmids produced about 20 times more hemolysin activity than the parental plasmid pHly152, when grown in minimal medium supplemented with hemoglobin. Thus high hemolytic activity is not lethal to the cells, contrary to previous assumptions.-hemolysin was purified from culture supernatants of strain SU100 (pSU157) by ammonium sulfate precipitation and gel filtration in Sephacryl S-200 in the presence of 6 M urea. When purified -hemolysin preparations were subjected to electrophoretic analysis in denaturing conditions, a single 107 kdal polypeptide was observed. This probably corresponds to the -hemolysin protein, since an isogenic E. coli strain carrying plasmid pSU161, an Hly^- mutant derivative of pSU157, did not synthesize the 107 kdal polypeptide.     

Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence

During senescence of primary bean leaves (Phaseolus vulgaris), there are differential changes in the rates at which thylakoid proteins are synthesized. In particular, synthesis of the 32 kD herbicide-binding protein continues throughout senescence, whereas formation of the and subunits of ATPase, the 68 kD photosystem I reaction center polypeptide, cytochrome f, cytochrome b₆ and the structural apoprotein of the lightharvesting chlorophyll protein complex (LHCP) declines. Pulse-chase experiments with intact leaves indicated rapid degradation of the 32 kD protein, which is consistent with its known rapid rate of turnover. This degradation was light-dependent and inhibited by DCMU, and the kinetics of degradation were similar for young and senescent membranes. In Coomassie-stained gels, the 68 kD reaction center polypeptide of photosystem I, the and subunits of ATPase and the LHCP were the dominant proteins for all ages of membranes. Western blot analysis indicated that cytochrome f and cytochrome b₆ are selectively depleted during senescence. The data have been interpreted as indicating that translational disruptions in both the cytoplasmic and chloroplastic compartments may contribute to the decline in photosynthetic electron transport in the senescing leaf.