Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH,Zn Concentrations and Humic Acids Chemical Properties

The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic acids with zinc.     

beta-Galactosidases in Ripening Tomatoes

Tomatoes (Lycopersicon esculentum L.) contained a high level of β-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of β-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, β-galactosidases I and III levels decreased but the β-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K_m value with p-nitrophenyl-β-galactoside, and stability with respect to pH and temperature. β-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of β-1, 4-linked galactose. The ability of β-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.     

Purification and properties of glucoamylase from sugar beet cells in suspension culture

Glucoamylase and α-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with α-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the α-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of α-mannosidase and α-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained α-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M_r (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K_m value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some α-amylases and the debranching enzyme.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

Rat small-intestinal β-galactosidases: Influence of pH on the hydrolysis of different substrates

1. The influence of pH and the kind of buffer on the hydrolysis of lactose and four hetero-β-galactosides (phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) by homogenates of rat small-intestinal mucosa has been studied. 2. There are at least two β-galactosidases present in the homogenates, one with optimum pH3–4 and another with optimum pH5–6. 3. The enzyme with the lower pH optimum is mainly a heterogalactosidase. It hydrolyses lactose slowly. The other enzyme is mainly a disaccharidase, since it hydrolyses lactose much more rapidly than the heterogalactosides. 4. Under the conditions used, citrate had an inhibitory effect on the 6-bromo-2-naphthyl β-galactosidase activity at pH3–4, but did not influence the 6-bromo-2-naphthyl β-galactosidase activity at pH5–6 or the hydrolysis of the other substrates at any pH.     

Crystal Structure of OXA-58 with the Substrate-Binding Cleft in a Closed State: Insights into the Mobility and Stability of the OXA-58 Structure

OXA-58 is a class D β-lactamase from the multi-drug resistant Acinetobacter baumannii. We determined the crystal structure of OXA-58 in a novel crystal, and revealed the structure of the substrate-binding cleft in a closed state, distinct from a previously reported OXA-58 crystal structure with the binding cleft in an open state. In the closed state, the movement of three loops (α3–α4, β6–β7, and β8–α10) forms an arch-like architecture over the binding cleft through interaction between the Phe113 residues of α3–α4 and Met225 of β6–β7. This structure suggests the involvement of these flexible loops in OXA-58 substrate binding. In contrast to the mobile loops, the Ω-loop appeared static, including the conserved loop residues and their hydrogen bonds; the pivotal residue Trp169 within the Ω-loop, ζ-carbamic acid of the modified base catalyst residue Lys86, and nucleophilic residue Ser83. The stability of OXA-58 was enhanced concomitant with an increase in the hydrolytic activity catalyzed by NaHCO₃-dependent ζ-carbamic acid formation, with an EC₅₀ of 0.34 mM. The W169A mutant enzyme was significantly thermally unstable even in the presence of 100 mM NaHCO₃, whereas the S83A mutant was stabilized with NaHCO₃-dependent activation. The ζ-carbamic acid was shown to increase not only OXA-58 hydrolytic activity but also OXA-58 stability through the formation of a hydrogen bond network connected to the Ω-loop with Ser83 and Trp169. Thus, the static Ω-loop is important for OXA-58 stability, whereas the mobile loops of the substrate-binding cleft form the basis for accommodation of the various substituents of β-lactam backbone.     

The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to be Regulated by the β4 Integrin Subunit

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH₂ terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH₂ terminus of the cytoplasmic domain of BP180.

To investigate the effect of the α6β4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack α6β4 and BP180, with cDNAs for BP180 as well as for human α6A and β4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with α6β4. In contrast, when cells were transfected with cDNAs for α6A and mutant forms of β4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with α6β4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for α6A and a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of the β1 integrin subunit, BP180 was not colocalized with the α6β4/β1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of β4 determine the subcellular distribution of BP180.

     

Stimulation of adenine phosphoribosyltransferase by adenosine triphosphate and other nucleoside triphosphates

1. Adenine phosphoribosyltransferase was protected from inactivation on heating at 55° by the presence of 5-phosphoribosyl pyrophosphate. ATP, adenine, AMP or GMP had no protective effect on the activity of this enzyme. The presence of either 5-phosphoribosyl pyrophosphate or ATP did not protect adenine phosphoribosyltransferase against the loss of ATP stimulation obtained by heating at 55°. 2. At pH5·3 and 6·0 adenine phosphoribosyltransferase was stimulated by a narrow range of ATP concentration (15–25μm). At pH6·5 and 7·0 maximum stimulation was obtained with 25–30μm-ATP, and at pH7·4, 8·2 and 8·85 maximum stimulation was obtained over a wide range of ATP concentrations (60–200μm). With extracts that had been heated for 30min. at 55° no stimulation was observed at either pH5·3 or 7·4 with ATP concentrations up to 100μm. 3. Short periods of heating at 55° (1, 2 or 5min.) increased the stimulation of adenine phosphoribosyltransferase obtained with various concentrations of ATP. 4. The addition of CTP, GTP, deoxy-GTP, deoxy-TTP or XTP to assay mixtures resulted in weak stimulation of adenine-phosphoribosyltransferase activity. 5. It is suggested that there are at least three different forms of adenine phosphoribosyltransferase, each with a different affinity for ATP.     

pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)₃, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)₃, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)₃, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)₃; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

A Neutral Thermostable β-1,4-Glucanase from Humicola insolens Y1 with Potential for Applications in Various Industries

We cloned a new glycoside hydrolase family 6 gene, Hicel6C, from the thermophilic fungus Humicola insolens Y1 and expressed it in Pichia pastoris. Using barley β-glucan as a substrate, recombinant HiCel6C protein exhibited neutral pH (6.5) and high temperature (70°C) optima. Distinct from most reported acidic fungal endo-β-1,4-glucanases, HiCel6C was alkali-tolerant, retaining greater than 98.0, 61.2, and 27.6% of peak activity at pH 8.0, 9.0, and 10.0, respectively, and exhibited good stability over a wide pH range (pH 5.0−11.0) and at temperatures up to 60°C. The K _m and V _max values of HiCel6C for barley β-glucan were 1.29 mg/mL and 752 μmol/min·mg, respectively. HiCel6C was strictly specific for the β-1,4-glucoside linkage exhibiting activity toward barley β-glucan, lichenan, and carboxy methylcellulose sodium salt (CMC-Na), but not toward laminarin (1,3-β-glucan). HiCel6C cleaved the internal glycosidic linkages of cellooligosaccharides randomly and thus represents an endo-cleaving enzyme. The predominant product of polysaccharide hydrolysis by HiCel6C was cellobiose, suggesting that it functions by an endo-processive mechanism. The favorable properties of HiCel6C make it a good candidate for basic research and for applications in the textile and brewing industries.     

Lysis of Yeast Cell Walls: Glucanases from Bacillus circulans WL-12

Endo-β-(1 → 3)- and endo-β-(1 → 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-β-(1 → 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-β-(1 → 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-β-(1 → 3)-glucanase was specific for the β-(1 → 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. β-(1 → 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-δ-lactone was a noncompetitive inhibitor and Hg²⁺ inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified β-(1 → 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified β-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.     

Cell Wall and Cytoplasmic Isozymes of Radish beta-Fructosidase Have Different N-Linked Oligosaccharides

When 36-hour-old dark grown radish seedlings are transferred to far-red light, there is a decrease in cytoplasmic β-fructosidase (βF) and an increase in cell wall βF compared to the dark controls. Cytoplasmic and cell wall-bound β-fructosidase are both glycoproteins and exhibit high antigenic similarities, but differ according to charge heterogeneity and carbohydrate microheterogeneity. Growth of radish seedlings in the presence of tunicamycin results in a partial inhibition of βF glycosylation but nonglycosylated βF still accumulates in the cell wall under far-red light. Thus, glycosylation is not necessary for intracellular transport, for correct targetting, or for wall association of an active βF. The nonglycosylated cytoplasmic and cell wall βF forms have the same relative molecular mass but glycosylated forms have different oligosaccharide side-chains, with respect to size and susceptibility to α-mannosidase and endoglycosidase D digestion. The oligosaccharides of both forms are partly removed by endoglycosidase H when βF is denatured. Isoelectric focusing analysis of βF shows that the cell wall-associated isozymes are more basic than the cytoplasmic isozymes, and that the charge heterogeneity also exists within a single plant. A time course of changes in βF zymograms shows a far red light stimulation of the appearance of the basic forms of the enzyme. However, the more basic cell wall specific βF forms are not present when N-glycosylation is prevented with tunicamycin. These results indicate that cytoplasmic and cell wall βF probably have common precursor polypeptides and basic cell wall forms arise via processing events which are tunicamycin sensitive.     

Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins : Evidence for the Inactivation of 6'-Hydroxyamatoxins

Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, α-amanitin, 6′-deoxy-α-amanitin, and 6′-O-methyl-α-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and α-amanitin was much less effective than the other two amatoxins, even though K_i values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of α-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the α-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role.     

Rat-kidney lysosomes: isolation and properties

1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of α-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate α-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal α-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6·2. 4. The stability of lysosomal suspensions was studied. Incubation at 37° and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the α-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6–7 and less stable at pH 8–9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0·6m to 0·25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.     

Characteristics of Two Forms of α-Amylases and Structural Implication

Complete (Ba-L) and truncated (Ba-S) forms of α-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.     

A STUDY OF SIEVE ELEMENT STARCH USING SEQUENTIAL ENZYMATIC DIGESTION AND ELECTRON MICROSCOPY

The fine structure of plastids and their starch deposits in differentiating sieve elements was studied in bean (Phaseolus vulgaris L.). Ultrastructural cytochemistry employing two carbohydrases specific for different linkages was then used to compare the chemical nature of "sieve tube starch" (the starch deposited in sieve elements) with that of the ordinary starch of other cell types. Hypocotyl tissue from seedlings was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon-Araldite. Treatment of thin sections on uncoated copper grids with α-amylase or diastase at pH 6.8 to cleave α-(1 → 4) bonds resulted in digestion of ordinary starch grains but not sieve element grains, as determined by electron microscopy. Since α-(1 → 6) branch points in amylopectin-type starches make the adjacent α-(1 → 4) linkages somewhat resistant to hydrolysis by α-amylase, other sections mounted on bare copper or gold grids were treated with pullulanase (a bacterial α-[1 → 6] glucosidase) prior to digestion with diastase. Pullulanase did not digest sieve element starch, but rendered the starch digestible subsequently by α-amylase. Diastase followed by pullulanase did not result in digestion. The results provide evidence that sieve element starch is composed of highly branched molecules with numerous α-(1 → 6) linkages.     

QUANTITATIVE ESTIMATION OF LYO- AND DESMOENZYMES IN TISSUE SECTIONS WITH AND WITHOUT FIXATION

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, β-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, β-glucuronidase, and arylsulfatase, ⅓, 2/3, 2/3, ½, ⅛, and ⅛ of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.     

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The ¹H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n₅₀), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P₅₀) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q), and rHb (βE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).