Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

Chloroplast and Mitochondrial DNA Variation in HORDEUM VULGARE and HORDEUM SPONTANEUM

A survey of restriction fragment polymorphism in Hordeum vulgare and Hordeum spontaneum was made using 17 and 16 hexanucleotide restriction endonucleases on chloroplast (cp) and mitochondrial (mt) DNA, respectively. The plant accessions originated from various places throughout the Fertile Cresent and Mediterranean. The types of changes in cpDNA consisted of nucleotide substitutions and insertions and deletions on the order of 100 base pairs. In contrast, mtDNA has most likely undergone larger insertions and deletions of up to 20 kilobase pairs in addition to rearrangements. Grouping of mtDNA fragment data showed that in some cases geographical affinities existed between the two species, whereas in others there were no clear affinities. Nucleotide diversity estimates derived from the restriction fragment data were used in a number of comparisons of variability. Comparisons of overall mtDNA variability (nucleotide diversity = 9.68 x 10^-4) with cpDNA variability (nucleotide diversity = 6.38 x 10^-4 ) indicated that the former are somewhat more variable. Furthermore, there was no indication that the wild H. spontaneum (cpDNA diversity = 5.57 x 10^-4; mtDNA diversity = 6.04 x 10 ^-4) was more variable than the land races of H. vulgare (cpDNA diversity = 5.88 x 10^-4; mtDNA diversity = 9.79 x 10^-4). In fact, on the basis of mtDNA diversity, H. vulgare was the more variable species. Comparison of organelle nucleotide diversity estimates with an estimate of nuclear nucleotide diversity derived from existing isozyme data provided evidence that both organelle genomes are evolving at a slower rate than the nuclear genome.     

Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Analyse von zwei Legumin-Genfamilien der Ackerbohne (Vicia faba L.)

DNA clones representing two subfamilies A and B of legum in genes and a recombinant phage containing a complete legumin B gene have been isolated and characterized by DNA sequencing. A DNA fragment harbouring the legumin B gene and adjacent sequences was used for Ti-mediated transfer into tobacco cells.     

G-11 staining in Turner's syndrome with mos 45,X/46,X,r(?)

Mos 45,X/46,X,r(?) in 4 patients with Turner's syndrome and no signs of virilization, and in one pair of monozygotic twins, one of them with clitoral hypertrophy, was studied using combined cytogenetic techniques and specially G-11 staining for the characterization of the X or Y origin of the rings. In all 6 patients the ring was G-11 positive, attesting its Y origin. Both twins were operated and bilateral streak gonads with a bilateral nodule of testicular tissue were found. Similar small rings were also studied in one patient with mos 46,XX/46,X,r(X) and in one nonvirilized Turner's syndrome patient with a larger ring; in these two cases the ring was G-11 negative. It seems that the small rings occasionally found in Turner's syndrome are more frequently from Y origin and therefore prophylactic gonadectomy should be considered.     

Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels

Survival of bacteria (Rhizobium, Agrobacterium, and Arthrobacter spp.), fungal spores (Penicillium sp.), and yeasts (Saccharomyces sp.) was studied in relation to water activity (a_w) and the presence of nutritive solutes. The cells were entrapped in polysaccharide gels, as is done to immobilize cells or enzymes, and then dehydrated. The number of living cells (10¹⁰ g of dry polymer⁻¹) remained constant for periods of storage of >3 years at 28°C when the inocula were kept at an a_w of <0.069. At a_w values between 0.069 and 0.83 the number of survivors diminished more and more rapidly as the a_w was raised. For a given a_w and organism, there were large differences in survival rate as a function of the nutritive solutes used to culture the microorganisms. Low-molecular-weight compounds (with three or five carbon atoms) had a deleterious effect on survival, whereas compounds of higher molecular weight (C₆ to C₁₂) had a protecting effect. Thus, the a_w alone was not a sufficient explanation for the deterioration of the inocula. Survival seemed to be more directly related to some properties of the water in the biopolymer. New concepts such as the discontinuity of properties of water and the point of mobilization of solutes, already proposed by Duckworth and Kelly (J. Food Technol. 8:105-113, 1973) and Seow (J. Sci. Food Agric., 26:535-536, 1975), have been taken into consideration to explain the interactions of water with the biopolymer and their specific effects on the microorganisms.     

Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA

Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.     

Plant growth regulation with triazoles of the dioxanyl type

The plant growth retarding activities of several dioxanylalkyl and dioxanylalkenyl triazoles were determined in seedlings of barley, rice, and oilseed rape. Out of these groups some substances proved to be among the most efficient growth retardants known. The compound 1-(4-trifluormethyl)-2-(1,2,4-triazolyl-(1))-3-(5-methyl-1,3-dioxan-5-yl)-propen-3-ol was investigated more closely. Shoot growth is reduced more intensively than root growth by this compound. At lower dosages root growth may even be stimulated. The action of this retardant can be antagonized by gibberellin A₃ and byent-kaurenoic acid. It is suggested that its main biochemical action is to block the reactions that lead froment-kaurene toent-kaurenoic acid in the course of gibberellin biosynthesis.     

Concordance of experimentally mapped or predicted Z-DNA sites with positions of selected alternating purine-pyrimidine tracts.

The recent electronmicroscopic and biochemical mapping of Z-DNA sites in phi X174, SV40, pBR322 and PM2 DNAs has been used to determine two sets of criteria for identification of potential Z-DNA sequences in natural DNA genomes. The prediction of potential Z-DNA tracts and corresponding statistical analysis of their occurrence have been made on a sample of 14 DNA genomes. Alternating purine and pyrimidine tracts longer than 5 base pairs in length and their clusters (quasi alternating fragments) in the 14 genomes studied are under-represented compared to the expectation from corresponding random sequences. The fragments [d(G X C)]n and [d(C X G)]n (n greater than or equal to 3) in general do not occur in circular DNA genomes and are under-represented in the linear DNAs of phages lambda and T7, whereas in linear genomes of adenoviruses they are strongly over-represented. With minor exceptions, potential Z-DNA sites are also under-represented compared to random sequences. In the 14 genomes studied, predicted Z-DNA tracts occur in non-coding as well as in protein coding regions. The predicted Z-DNA sites in phi X174, SV40, pBR322 and PM2 correspond well with those mapped experimentally. A complete listing together with a compact graphical representation of alternating purine-pyrimidine fragments and their Z-forming potential are presented.