Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium

Summary Clostridium stercorarium cultures grown on cellobiose contain both an extracellular and a cell-bound -glucosidase activity. A substantial portion of the cell-bound enzyme could be extracted by osmotic shock, suggesting a periplasmic localization. The -glucosidase present in culture supernatants was purified to homogeneity. It was found to be identical in all aspects tested with the cell-bound -glucosidase. The enzyme exists as a monomer with an apparent molecular weight of 85.000 (SDS-PAGE) and a pI of 4.8. It shows optimal activity as pH 5.5 and 65° C. Thiol groups are essential for enzyme activity. In the presence of reducing agents and divalent cations the half-life of the purified enzyme was more than 5 h at 60°C. The enzyme hydrolyses at different rates a wide range of substrates including aryl--glucosides, cellobiose, and disordered cellulose. K _m values were determined as 0.8 mM for p-nitrophenyl--glucoside (PNPG) and 33 mM for cellobiose. The cellular localization and the substrate specificity pattern are consistent with a dual role of the C. stercorarium -glucosidase in cellulose saccharification: (1) Cleavage of cellobiose formed by exoglucanase and (2) degradation of cellodextrins produced by endoglucanase action.     

The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation,stoichiometry, reassembly and topology of individual subunits

Summary Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termed, and, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunit is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termed. A fifth, diffuse component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies of and 8 copies of/ within the 2×6 particles. The: ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura. exists in 2 copies inAstacus andCancer which means that a single dimer- is present in a two-hexamer. This leaves 2 monomeric copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacus - was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (F_ab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridge- is surrounded by two and two subunits forming the central core of the dodecamer. Two additional and two additional subunits form the periphery together with one subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This paper is decicated to Professor Dr. Bernt LinzenPreliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.     

Alterations in the regulatory region of the human papillomavirus type 6 genome are generated during propagation in Escherichia coli.

We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli. Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence. Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion. Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp. One plasmid contained a single insertion of 77 bp. The 94-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths. All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400). Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones. We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E. coli and that the alterations are the result of recA-independent events. These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion. In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical. Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR.     

Evidence for a non-opioid sigma binding site in the guinea-pig myenteric plexus

The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site (KD = 46 +/- 5 nM; Bmax = 3.4 +/- 0.5 pmole/g wet weight) and a low affinity site (KD= = 342 +/- 72 nM; Bmax = 22 +/- 3 pmole/g wet weight). Morphine and naloxone 10(-4) M were unable to displace (+)-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Effect of CO2 enrichment and nitrogen availability on resource acquisition and resource allocation in a grass,Bromus mollis

Summary The effects of CO₂ enrichment on the growth, biomass partitioning, photosynthetic rates, and leaf nitrogen concentration of a grass, Bromus mollis (C₃), were investigated at a favorable and a low level of nitrogen availability. Despite increases in root: shoot ratios, leaf nitrogen concentrations were decreased under CO₂ enrichment at both nitrogen levels. For the low-nitrogen treatment, this resulted in lower photosynthetic rates measured at 650 l/l for the CO₂-enriched plants, compared to photosynthetic rates measured at 350 l/l for the non-enriched plants. At higher nitrogen availability, photosynthetic rates of plants grown and measured at 650 l/l were greater than photosynthetic rates of the non-enriched plants measured at 350 l/l. Water use efficiency, however, was increased in enriched plants at both nitrogen levels. CO₂ enrichment stimulated vegetative growth at both high and low nitrogen during most of the vegetative growth phase but, at the end of the experiment, total biomass of the high and low CO₂ treatments did not differ for plants grown at low nitrogen availability. While not statistically significant, CO₂ tended to stimulate seed production at high nitrogen and to decrease it at low nitrogen.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance