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Magnesium-deficient patients show dysfunctional calcium (Ca(2+)) metabolism due to defective parathyroid hormone (PTH) secretion. In mice and rats, long-term magnesium (Mg(2+)) deprivation causes hyperphosphaturia and increases fibroblast growth factor 23 (FGF23) secretion, despite normal serum phosphate (Pi) and Ca(2+). Electrolyte disturbances during early hypomagnesemia may explain the response of mice to long-term Mg(2+) deprivation, but our knowledge of electrolyte homeostasis during this stage is limited. This study compares the effect of both short- and long-term Mg(2+) restriction on the electrolyte balance in mice. Mice were fed control or Mg(2+)-deficient diets for one to three days, one week, or three weeks. Prior to killing the mice, urine was collected over 24 h using metabolic cages. Within 24 h of Mg(2+) deprivation, hypomagnesemia, hypocalcemia and hyperphosphatemia developed, and after three days of Mg(2+) deprivation, serum potassium (K(+)) was increased. These changes were accompanied by a reduction in urinary volume, hyperphosphaturia, hypocalciuria and decreased Mg(2+), sodium (Na(+)) and K(+) excretion. Surprisingly, after one week of Mg(2+) deprivation, serum K(+), Pi and Ca(2+) had normalized, showing that mineral homeostasis is most affected during early hypomagnesemia. Serum Pi and K(+) are known to stimulate secretion of FGF23 and aldosterone, which are usually elevated during Mg(2+) deficiency. Thus, the hyperphosphatemia and increased serum K(+) concentration observed during short-term Mg(2+) deprivation may help our understanding of adaptation to chronic Mg(2+) deficiency.

Corrosion behavior of zinc coatings produced from ammonium chloride + zinc chloride + potassium chloride bath (ACB) and zinc chloride + potassium chloride + boric acid bath (BAB) was studied using weight-loss and electrochemical techniques. It was established that the zinc coatings electrodeposited in ACB performs better than the electrodeposited coatings produced from BAB. The zinc deposition reactions in baths having ammonium chloride polarized to a greater extent than the bath blended with boric acid. Alternating current (AC) impedance studies demonstrated that the coatings produced in both types of solutions formed unstable film in corrosive media. This was corroborated by weight-loss as well as direct current (DC) polarization techniques. The presence of boric acid in the plating bath had little influence on charge-transfer resistance of zinc deposition. However, the electrochemical double-layer capacitance decreased with the increase of boric acid concentration in the bath. Scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) studies revealed that the coating produced in ACB has amorphous-type grains whereas the BAB coating showed a crystalline structure. It was concluded that improved corrosion resistance of the zinc coating deposited in the ACB was caused by the stronger complexing tendency of ammonium chloride with zinc cations, which resulted in a slow discharge of this ion during the deposition process.

In the present study, the effects of orally-administered lithium on testicular morphology were examined in the spotted munia (Lonchura punctulata), a seasonally breeding sub-tropical finch. Adult males were procured from natural populations during the month of August, a time when these birds begin to show seasonal reproductive maturity in an annual cycle. Both during the period of acclimation, and throughout the subsequent experimental period, the birds were maintained in an open aviary simulating natural environmental conditions. Lithium was dissolved in distilled water and was administered via the oral route by means of a commercially available stomach-tube. A total of five experimental groups were utilized. The first group (Group A) served as control and received lithium-free distilled water in a similar manner. In the remaining four groups, lithium was administered daily as follows: Group B (2.5 mEq/Kg body weight for 5 days); Group C (2.5 mEq/Kg for 10 days); Group D (5.0 mEq/Kg for 5 days) and Group E (5.0 mEq/Kg for 10 days). All lithium administrations were carried out between 14:00 and 15:00h. Twenty-four hours after the last oral lithium, final body weights were recorded, blood samples were obtained (by brachial vein puncture for the measurement of serum lithium) and the animals were sacrificed, and testes were collected for histological studies. Our results indicated that lithium treatment led to a significant reduction in testicular weight and seminiferous tubular diameter, and a marked degenerative changes in germ cells in that most of the spermatids and mature spermatozoa showed necrotic changes and were sloughed off from the seminiferous tubular epithelium. Complete desquamation and loss of germ cells, and their clump formation were also noted within many seminiferous tubular lumen. Notably these adverse effects were observed when serum lithium levels were within the therapeutic range for human. These results confirm our earlier report on lithium's adverse effects on testicular function, and extend further to show that lithium indeed has a significant adverse effect on the histomorphology, and, thus, the function of the testis in birds.

Arsenic contaminated waters are not uncommon; indeed from naturally occurring contaminated waters through to those that are a direct consequence of human activities such as mining, all are affecting the quality of water resources worldwide. The ever increasing demands on natural water resources mean that the effective control of this toxic contaminant is paramount and this is reflected in the ever increasing global legislation. There are currently three mechanisms by which arsenic is commercially treated in effluents. These are physical separation processes such as reverse osmosis, precipitation/adsorption processes, some of which are bacterially assisted, and a whole variety of ion exchange processes, again with some bacterial enhancement. The choice of treatment is not only driven by cost but by the chemistry of the water and the water quality standard to be met. In this study a very high arsenic enriched groundwater, containing in excess of 25,000 µg/L arsenic, was treated by a typical treatment method through a continuously operated pilot plant. In the treatment, iron III salts were added to the influent in order to form precipitates with the arsenic and to form an adsorptive surface that would assist with treatment of the enriched water. This addition of iron III salts for the removal of arsenic is common practice in the water treatment industry as the resulting iron III arsenates are highly stable. However, results from the pilot plant show that the process was further enhanced by the addition of small amounts of hydrogen peroxide. Hydrogen peroxide is a powerful oxidising reagent and assists in ensuring the complete conversion of any arsenic III to arsenic V that was then effectively removed in the pilot plant. After treatment residual arsenic levels of 10 µg/L were obtained compared to 68 µg/L without oxidation reagent addition.

Recent basic and clinical advances have consolidated the concept of tissue-selective estrogens, i.e. molecules that express different degrees of partial agonist, full agonist or antagonist activity in different tissues or cells. Delta8,9-Dehydroestrone sulfate (delta8,9-DHES) is a conjugated estrogen and a component of conjugated equine estrogens (CEE). It is metabolized in the human in at least a 1:1 ratio to its 17beta form, 17beta-delta8,9-DHES. To evaluate its activity in different clinical and biochemical parameters, a clinical research study was conducted with delta8,9-DHES and estrone sulfate as a comparator in postmenopausal women. Delta8,9-DHES was given orally at a daily dose of 0.125 mg for 12 weeks in a group of 10 women. Two additional groups of women received either estrone sulfate alone (1.25 mg/day) or the combination of delta8,9-DHES and estrone sulfate at the previously specified doses. A significant and consistent suppression of hot flushes (number, severity, and total score) was observed with delta8,9-DHES, reaching more than 95% suppression in all parameters of vasomotor symptoms. This level of activity was equal to that obtained with the much higher dose of estrone sulfate, and it was sustained for the duration of the treatment period (12 weeks). Measurements of a bone resorption marker, i.e. urinary excretion of N-telopeptide, demonstrated that delta8,9-DHES at 8 weeks produced a degree of suppression (40%) similar to that observed with the higher dose of estrone sulfate. Gonadotropin secretion (FSH and LH) was significantly suppressed in women receiving delta8,9-DHES, similar to that observed with estrone sulfate alone or with the combination of the two. Other parameters, such as total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol were not modified significantly, whereas serum globulins (sex hormone-binding globulin and corticosteroid-binding globulin) showed only marginal increases after delta8,9-DHES administration. Taken together with preclinical data, it is found that delta8,9-DHES is an active estrogen with a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine (gonadotropin and PRL) and bone preservation parameters, whereas displaying little or no efficacy, at the dose tested, on other peripheral parameters normally affected by estrogens. Collectively, this information supports the concept that delta8,9-DHES is an integral component of CEE, with distinct tissue selectivity contributing to the CEE's overall clinical activity, and places this estrogen as a distinct member of a novel class of centrally active molecules with unique peripheral tissue selectivity.

The accumulation of specific soluble carbohydrates has been implicated in the acquisition of desiccation tolerance and improved longevity in orthodox seeds, leading to the hypothesis that carbohydrate composition might be used as a diagnostic marker for seed storage category. We have studied the sugar composition of 46 tissues from seeds of 18 species, covering 13 genera and ten families, and representing three seed storage categories: orthodox, intermediate and recalcitrant. Soluble carbohydrate extracts from each tissue were analysed using High pH Anion Exchange Chromatography. Total sugar content and sucrose level of the embryo were highly variable across all species and no simple association with seed storage physiology was evident. Monosaccharide levels were found to be low in most seeds studied, including those of the recalcitrant category. Sucrosyl-oligosaccharides, raffinose and stachyose, were observed to be lower in recalcitrant seeds compared to orthodox seeds. In general, orthodox and recalcitrant seeds had tissues with sucrosyl-oligosaccharide:sucrose mass ratios of>0.143 (i.e. 1:7) and

The central benzodiazepine receptor tracer [ N -methyl- 11 C]iomazenil (Ro 16-0154) was synthesized by alkylation of the desmethyl precursor noriomazenil with [ 11 C]methyl iodide. The [ 11 C]CH 3 I (prepared by reduction of [ 11 C]CO 2 with LiAlH 4 followed by reaction with HI) was reacted with noriomazenil in N,N -dimethylformamide and Bu 4 N + OH − for 1 min at 80 °C and purified by HPLC (C 18 , 34% CH 3 CN/H 2 O, 7 mL/min). The product was obtained with synthesis time 35 ± 5 min (mean ± SD, n = 7), radiochemical yield (EOB) 36 ± 16%, radiochemical purity 99 ± 1%, and specific activity 5100 ± 2800 mCi/μmol. Absorbed radiation doses were calculated from previously acquired human biodistribution data. The urinary bladder wall received the highest dose (0.099 mGy/MBq) for 4.8 h voiding interval and the effective dose equivalent was 0.015 mSv/MBq. After i.v. injection of [ 11 C]iomazenil in an adult baboon or healthy human volunteer, radioactivity accumulated in the cortex with time-activity curves in agreement with results obtained with [ 11 C]flumazenil PET and [ 123 I]iomazenil SPECT studies. The count rate was sufficient to obtain quantitative images up to 2 h post-injection with a 14mCi injection. These results suggest that [ 11 C]iomazenil will be a useful agent for measuring benzodiazepine receptors in vivo by positron emission tomography.

Incubation of intact washed rapidly growing tomato (hybrid of Lycopersicon esculentum and L. peruvianum) suspension cells with 0.2 M phosphate buffer pH 7.0 for 23 hr at 25° liberated a significant amount of oxidizing agent and a selective inhibitor of peroxidase-mediated cross-linking of extensin precursor in vitro. Further studies revealed that an endogenous heat-stable oxdizing agent and heat-labile inhibitor are two separate low molecular weight components (Mr < 5000) and that the oxidizing agent is not H2O2.

[1] Biosphere-atmosphere exchange of NOx at the Sundarban mangrove forest along the northeast coast of the Bay of Bengal, India, showed uptake rates of −0.84 to −1.63 ng N m−2 s−1 during the day and both uptake and emission rates of −0.36 to 5.19 ng N m−2 s−1 during the night from September to February. However, during the period from March to August, NOx emission ranged between 0.34 and 2.13 ng N m−2 s−1 and 0.88 and 3.26 ng N m−2 s−1 in daytime and nighttime, respectively. During the postmonsoon period, NOx uptake could be attributed to mangrove stomatal activity during the day. Mangroves absorbed nitrogen from both the soil and the atmosphere. Seasonal and diurnal variability of NOx and O3 is partly due to plant growth in the postmonsoon period. In addition to the NOx-O3 photochemical cycle, stomatal uptake of NOx could also be an important process for keeping a low-ozone state at the land-ocean boundary of the northeast coast of the Bay of Bengal.

Low temperature, molecular beams of polyenes (retinol, retinal, a Schiff base retinal, and β‐carotene) are formed in jet expansions by CO2 laser‐induced desorption from a deposited solid surface. Mass spectra of these intact polyenes are detected by resonantly‐enhanced ionization mass spectrometry. Laser‐desorption conditions which produce essentially no polyene fragmentation are obtained by controlling the focusing conditions of the CO2 laser on the deposited sample.

This study aims to investigate the control of arsenic distribution by biogeochemical processes in the Indian Sundarban mangrove ecosystem and the importance of this ecosystem as an arsenic source for surrounding coastal water. The As(V)/As(III) ratio was found to be significantly lower in both surface and pore waters compared to sea water, which could be attributed to biogeochemical interconversion of these arsenic forms. The biological uptake of arsenic due to primary and benthic production occurs during the post-monsoon season, and is followed by the release of arsenic during the biochemical degradation and dissolution of plankton in the pre-monsoon season. These results suggest that arsenic is immobilized during incorporation into the arsenic-bearing initial phase, and unlikely to be released into pore water until the complete microbial degradation of arsenic-bearing organic compounds.

The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457–469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40–50 nm in length and 2.0–2.5 nm in width. A minority of these particles showed a central ‘kink’. A number of smaller and generally wider particles (20 nm×2–4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer–dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 µM and sedimentation velocity data generated a Kd between 1 and 10 µM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of β-l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)4 — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.

Pigeonpea ( Cajanus cajan ) urease was immobilized on 1 cmx1 cm DEAE-cellulose paper strips. The optimum immobilization (51% activity) was observed at 4 degrees C, with a protein concentration of 1.0 mg/strip. The apparent optimum pH shifted from 7.3 to 6.8. Immobilized urease showed an optimal stability temperature of 67 degrees C, compared with 47 degrees C for the soluble urease. Time-dependent kinetics of the thermal inactivation of the immobilized urease were examined and found to be monophasic as compared with the soluble enzyme, which was biphasic. The Michaelis constant ( K (m)) for the DEAE-cellulose-immobilized urease was found to be 4.75 mM, 1.5 times higher than the soluble enzyme. Immobilized strips stored at 4 degrees C showed an increased half-life ( t (1/2)=150 days). There was practically no leaching of the enzyme from the immobilized strips over a period of 2 weeks. These strips were used for estimating the urea content of blood samples; the results obtained matched well with those obtained in a clinical laboratory through an Autoanalyzer(R) (Zydus Co., Rome, Italy). The easy availability of pigeonpea urease, the ease of its immobilization on DEAE-cellulose strips and the significantly lower cost of urease described in the present study makes it a suitable product for future applications in diagnostics.

alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), an enzyme responsible for mobilizing the raffinose family of oligosaccharides in legume seeds, has been isolated from lentils (Lens culinaris) and purified about 4,000-fold. The Sephadex gel filtration profile showed the presence of two forms of the enzyme, alpha-galactosidase I with an apparent Mr = 160,000 and alpha-galactosidase II of Mr = 40,000. Enzyme II readily aggregates to form I when any attempt is made to concentrate the solution. Thus, only enzyme I was purified and its properties studied. The multistep purification procedure included affinity binding of the enzyme to concanavalin A-Sepharose, indicating its glycoprotein nature with glucose/mannose termini of the carbohydrate moieties. The amino acid and carbohydrate compositions of the native enzyme and that of the glycopeptide obtained from pronase-digested, denatured enzyme have been determined. Asparagine seems to be involved in forming the linkage with the carbohydrate moiety. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme I shows a single protein band with Mr = 40,000 which also stains with periodic acid-Schiff reagent. Thus, the enzyme consists of four identical glycoprotein subunits. The isoelectric point of the enzyme is 8.0. The pH optima of enzyme I and II are 6.1 and 4.7, respectively. The substrate specificity and the mode of substrate inhibition of enzyme I is discussed. The effect of temperature on Vmax and Km of the enzyme is presented; at pH 6.1 the energy of activation is 62.1 kJ/mol and the delta H value is -34.3 kJ/mol in the temperature range 20-50 degrees C. Preliminary studies show that enzyme I possesses hemagglutinating properties with glucose/mannose specificity.

During ripening, mango mesocarp cell walls undergo degradation with the net loss of arabinose, galactose and galacturonic acid. Hot water fractions of the cell walls from unripe fruits were rich in galactose and arabinose and contained only 7% galacturonic acid in comparison with those from unripe fruits which contained 90% uronic acid: little change occurred in the alkali-soluble (hemicellulose) fraction during ripening. The ripening-associated changes in the cold water-soluble cytoplasmic polysaccharides in the mesocarp were also examined. As the mesocarp softened these increased in amount and bound uronic acid increased three-fold. Gel-filtration and ion-exchange chromatography were used to examine these cytoplasmic polysaccharides. Their average M r decreased on ripening and most of the fractions were complex with respect to monosaccharide composition. However, polysaccharides which are essentially an arabinoxylan and a galacturonan appeared to be present in the unripe and ripe tissue, respectively.

Sweet almond α-galactosidase (α- d -galactoside galactohydrolase, EC 3.2.1.22) catalyses hydrolytic, synthetic ( de novo ) and transfer reactions. Transfer products were formed using p -nitrophenyl α- d -galactoside as the galactosyl donor and glucose, galactose, sucrose, maltose and lactose as acceptors; several of the products were identified. The enzyme also caused elongation of the oligosaccharide chain of two substrates (melibiose and raffinose). In addition, the enzyme catalysed condensation of free galactose, yielding oligosaccharides. The products were identified in all cases by chromatography.

The amino acid composition of β- l -arabinosidase (ec 3.2.1.88) from Cajanus indicus seeds showed a high percentage of aspartic and glutamic acids. The isoelectric point was found to be 4.4. The effect of pH in the range 2.5–7.2 on Km was very small. However, variation of Vmax with pH indicated the presence of two ionizable groups with approx. pK values of 3 and 5.8. The groups were suspected to be carboxyl and histidyl-imidazolium, respectively. The presence of histidine at the active site was further strengthened by photo-oxidative inactivation of the enzyme and the protection from inactivation by the substrate. Destruction of the histidine residue was also demonstrated by amino acid analysis of the photo-oxidized enzyme. The enzyme displayed a transglycosylation property with retention of the anomeric configuration of the product. The effect of some nucleophiles on Km was negligible, while Vmax was increased. The effect of temperature on Vmax in the range 4–40°C showed a break-point at 18°C in the Arrhenius plot; the energies of activation below and above this point were 32 and 16 kcal·mol−1, respectively. At 4°C the liberation of aglycone residues of the substrate was stoichiometric with the enzyme concentration. Based upon the kinetic properties and transglycosylation results of the enzyme, a two-step mechanism of action was proposed which envisaged the formation of a glycosyl-enzyme intermediate.

The major sugars of fresh seeds of Castanea sativa were shown to be raffinose, stachyose and sucrose. Drying seeds at 25° for 14 weeks increased the ratio raffinose: stachyose from 1.1 to 3.5, reduced sucrose content by ca 50 % and decreased total extractable α-galactosidase. The enzyme activity was resolved into two peaks, a high MW form I (apparent MW215 000) and a low MW form II (apparent MW 53 000). The latter form was predominant in the extract of fresh seeds whereas the former was the main form in the 14-week dried seeds. An increase in the amount of enzyme I was also observed when a buffered extract (pH 5.5) of fresh seeds was stored at 4°. Enzymes I and II had pH optima of 4.5 and 6, respectively. Both enzymes hydrolysed p -nitrophenyl α- d -galactoside at a much greater rate than the natural substrates raffinose, stachyose, locust bean gum and carob gum. However, enzyme I showed preference for stachyose as compared to raffinose; the opposite order was observed for enzyme II.

The purification of α-galactosidase II 1 from mature Vicia faba seeds is reported. The enzyme ( M , 45 730 ± 3070) did not appear to be contaminated with the classical lectin favin: but possessed D -glucose/ D -mannose-specific lectin activity. Four D -mannose residues/mole enzyme bind with a K A of 2.18 x 10 3 M −1 and this binding is not inhibited by D -galactose. These observations, together with heat inactivation and pH studies, suggest that II 1 has different loci associated with its enzymic and lectin properties.

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N -acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - α-galactosidase I, an enzyme lectin from Vicia faba . The latter is shown to contain high mannose carbohydrate chains.

UDP-Galactose 4′-epimerase was purified ca 800-fold through a multi-step procedure which included affinity chromatography using NAD+ -Agarose. Three forms of the enzyme were separated by gel-filtration but only the major form was purified. The pH optimum of the enzyme was 9.5. Exogenous NAD+ was not required for enzymic activity but its removal caused inactivation. The enzyme was unstable below pH 7.0 but stable at pH 8.0 in the presence of glycerol and at −20° for two months. The equilibrium constant for the enzyme-catalysed reaction was 3.2 ± 0.15. The Km for UDP-galactose and UDP-glucose were 0.12 mM and 0.25 mM, respectively. The inhibition by NADH was competitive, with a Ki of 5 μM. The MW of the enzyme was 78 000; the two minor forms showed the values of 158 000 and 39 000, respectively.

The nature of the glucose/mannose specific lectin activity of α-galactosidase I from Vicia faba seeds has been examined. Gel filtration in the presence of high concentrations of glucose and SDS-PAGE failed to detect favin, a classical lectin which also occurs in the seed. A comparison of the haemagglutinating activities of the α-galactosidases from Vigna radiata and V. faba seeds strongly suggests that the catalytic site of the Vigna enzyme is also responsible for its agglutinating activity and that the catalytic and lectin sites are at different loci in the case of V. faba α-galactosidase I. The latter conclusion is supported by an investigation of the effects of glucose, mannose and galactose on the catalytic and lectin activities and by results obtained by demetallization of the V. faba enzyme. A single galactose-binding site and two mannose binding sites per subunit of enzyme I were detected by the method of equilibrium dialysis and the association constants for these monosaccharides measured. Mannose did not appear to affect the binding of galactose to the enzyme or vice versa . The removal of glycan chains from α-galactosidase I with endo-β- N -acetylglucosaminidase H released an active dimeric form of α-galactosidase. The possible involvement of lectin-glycoprotein interactions in the stabilization of the tetrameric form of the enzyme is considered.

Chalcone isomerase (EC 5.5.1.6) from cell suspension cultures of Phaseolus vulgaris has been purified about 400-fold. The molecular weight, as estimated by gel-filtration and SDS-polyacrylamide gel electrophoresis, is approx. 28 000. No isoenzymic forms are observed. The enzyme, which appears to require no cofactors, catalyses the isomerisation of both 6′-hydroxy and 6′-deoxy chalcones to the corresponding flavones. Likewise, a range of both 5-hydroxy and 5-deoxy flavonoids and isoflavonoids act as competitive inhibitors. The most potent inhibitors include the naturally occurring antimicrobial comcpounds kievitone ( K i 9.2 μ M) and coumestrol ( K i 2.5 μ M). The kinetics of the isomerisation of 2′,4,4′-trihydroxychalcone to the flavanone liquiritigenin have been investigated at a range of pH values. The pH optimum was around 8.0 and K m changed with pH in a manner consistent with control by groups which ionise with p K a values of 7.05 and 8.7 respectively. At pH 8.0, the energy of activation was 17.56 kJ/mol in the range 25–40°C. The role of the enzyme in the induced accumulation of flavonoid/isoflavonoid derivatives inthe Frech bean in discussed.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.

Two molecular forms, I and II (high and low molecular mass) of alpha-galactosidase were demonstrated in dry mung beans and a multi-step procedure was developed for isolating the tetrameric enzyme I in good yield. Two affinity chromatographic techniques were employed and an overall 10 000-fold purification was achieved. The enzyme was able to catalyse the hydrolysis of alpha-galactosidic linkages as well as agglutinate rabbit erythrocytes (clot formation). The clot was temporary and dissolved on longer incubation, yielding free galactose. The pH optima for both activities were similar. The enzyme also destroyed human-blood-group-B activity and increased H activity. The effect of pH on Km and Vmcx of the enzyme indicated the importance of carboxyl (pK approximately equal to 4.0) and histidine (pK approximately equal to 6.5) groups for activity. This was confirmed by amino acid modification experiments in the absence and presence of the substrate. The stoichiometry of enzyme inactivation showed the probable presence of 12 carboxyl groups and 9 histidine imidazole groups/molecule enzyme in the active site. The effects of modification of the groups on enzymic and hemagglutinating activities were parallel. A model explaining the display of both the activities by the enzyme, is presented in which hemagglutination is shown to be due to the formation of slightly stable enzyme-substrate complex. It is proposed that a true lectin should not alter the covalent status of the binding sugar.

The extractability of α-galactosidase activity from mature Vicia faba seeds and the conversion of the low molecular weight form II to the larger oligomer I, was examined over a range of salt concentrations. Specific and total activities of the preparations were high when strong salt solutions were used. Extraction of α-galactosidase I, in comparison with II, requires solutions with a high ionic strength (e.g. 0.5 M NaCl). Interpretation of gel filtration patterns are, however, complicated by conversion of II to I which occurs under these conditions. This conversion is also enhanced by routine procedures used for enzyme purification, such as citric acid precipitation.

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.

The presence of essential catalytic groups in sweet almond α-galactosidase (α- d -galactoside galactohydrolase, EC 3.2.1.22) has been investigated with the use of metal ions and photo-oxidation. Competitive inhibition was shown to occur with both Hg2+ and Ag+. From the inhibition data it was postulated that carboxyl and histidine groups in the enzyme active site were responsible for binding the metal ions. The latter group was destroyed by photo-oxidation resulting in inactivation of the enzyme. The enzyme was found to catalyse hydrolytic, transgalactosylation and synthetic reactions on the same active site. All the reactions proceeded yielding products with complete retention of configuration. Two possible mechanisms of action have been suggested and discussed.

A new enzyme, β- l -arabinosidase, has been detected in the dormant seeds of Cajanus indicus . This enzyme is different from α-galactosidase and does not hydrolyse an α- d -galactoside or an α- d -fucoside.

1. The hydrolysis of a variety of galactosides and other glycosides by α-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl α-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on Km and Vmax. values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

1. alpha-Galactosidase from sweet almonds was purified about 2000-fold through eight steps. 2. The enzyme preparation was free from other related enzymes known to occur in sweet almonds, and behaved as a homogeneous protein on filtration through Sephadex G-75. 3. A molecular weight of about 33000 was determined from the gel-filtration data. 4. The ultraviolet-absorption spectrum and thermal inactivation of the enzyme are described. 5. The purified enzyme hydrolysed p-nitrophenyl alpha-d-galactoside at a much faster rate than melibiose. 6. The pH optimum was at 5.5-5.7. 7. Besides hydrolysis, it also catalysed transfer of galactosyl residues, chain elongation of melibiose and the synthesis of oligosaccharides from galactose.

The effect of temperature and pH on the activity of sweet almond α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) has been studied with melibiose and β-nitrophenyl α- d -galactoside (PNPG). Activation energies for the two substrates were found to be 12.4 and 19.0 kcal/mole, respectively. The p K m vs. t /T plots were linear for both substrates, and the enthalpies of binding were −7.3 and −10.2 kcal/mole for PNPG and melibiose, respectively. Changes in K m and v max of melibiose with pH suggested the participation of a group with p K 5.75 in the enzymep-substrate binding and of another group with p K around 3 in the catalytic step. Similar studies with PNPG also revealed the presence of two groups ( p K 3.4 or above and 6.7 or below), the p K values of which were strongly influenced by the substrate concentration. The group dissociating in the lower pH range appears to play a minor part only in the PNPG hydrolysis. In spite of apparently marked differences in the effect of temperature and pH, all the substrates appear to be hydrolysed at the same site on the enzyme molecule. The differences probably arose from differences in the mechanisms of the reaction or in the nature of the rate-limiting step in the two cases.

α-Galactosidase from Vicia faba seeds has been resolved into three molecular forms, I, II 1 and II 2 , respectively. Enzyme I is a tetramer ( M r 160 000) consisting of identical sub-units ( M r 44000 ± 2000). All three forms display lectin activity with glucose/mannose specificity. Enzyme I has been further studied with respect to its lectin specificity and various factors affecting this property. The results indicate that the catalytic and the lectin sites reside in the same protein molecule. The results presented are unique in that the enzyme activity is specific for galactose and its lectin activity is specific for glucose/mannose.

High levels of ‘alkaline’ invertase activity occur in dormant mung bean seeds. During germination this activity decreases rapidly and is replaced by high ‘acid’ invertase activity. Cycloheximide prevented the formation of the latter activity and also inhibited germination. It is suggested that de novo synthesis of ‘acid’ invertase occurs during germination. Both enzymes bind to concanavalin A and, hence, are presumed to be glycoproteins. Affinity-purified enzyme samples show similar ratios of ‘acid’ and ‘alkaline’ invertase activities to the crude preparations indicating that specific enzyme inhibitors or activators are probably not involved in controlling the activities in vivo .

α-Galactosidase I from Vicia faba seeds binds to potato starch and sheep erythrocytes. With the aid of fluorescence microscopy and using 4-methylumbelliferyl α- D -galactoside as the substrate it has been demonstrated that the binding is via the lectin sites of the enzyme leaving catalytic sites free and detectable. The lectin site is specific for D -glucose/ D -mannose residues.

Tetrameric α-galactosidase I from Vicia faba seeds is dissociated with urea (2.5-5.0 M) into active sub-unit forms. At low urea concentrations dissociation is only apparent if methyl α-D-mannoside is present which may be indicative of the involvement of lectin interactions in subunit aggregation. Chromatofocusing of α-galactosidase I yields multiple tetrameric forms with pI values ranging from 8.75 to 7.35. It is suggested that native α-galactosidase I is a closely related mixture of tetramers resulting from post-translational changes in the enzyme protein.

Publisher Summary This chapter provides an overview of the biochemistry of α- D -Galactosidic linkages in the plant kingdom. Sucrose is a disaccharide that is most widespread amongst higher plants; its concentration varies from species to species. The metabolism of this disaccharide is well documented. Sucrose is formed as a major product of photosynthesis in higher plants and is generally the main form of translocate from leaves to other organs. It is a major carbohydrate-storage material that provides a ready source of D -glucose and D -fructose for the liberation of energy. Sucrose is also an important precursor for the synthesis of D -glucosyl esters of nucleoside diphosphates, and thus it takes part in the biosynthesis of complex oligosaccharides and polysaccharides. Combination of D -galactose with the D -glucosyl or D -fructosyl group of the sucrose molecule gives rise to a number of oligosaccharides; these are, generally, α- D -galactosides. The chapter discusses in detail about α- D -galactosides of sucrose. It also describes the α- D -galactosides of polyols and presents an overview of α- D -galactopyranosyl-specific lectins.

Publisher Summary From a physiological point of view, a fruit can be defined as the structural entity resulting from the development of the tissue that supports the ovule. The plant cell consists of cytoplasm surrounded by a cell wall. Each cell is connected to the adjacent cells by a pectin-rich middle lamella. Fruits are generally harvested at the mature stage when growth has ceased, and full development may be achieved independent of the parent plant with negligible impairment to quality. Fruits and their products constitute a commercially significant food commodity. The primary cell wall constituents can be divided into pectic polysaccharides (34%), hemicellulose (24%), cellulose (23%), and hydroxy proline-rich glycoprotein (19%). Pectic polysaccharides are generally considered to be those portions of cell walls that can be extracted by a variety of mild methods such as hot water, ammonium oxalate solution, weak acids, chelating agents, and endopolygalacturonase. The way in which the various components of the cell wall are linked together is largely not known. This is due mainly to the limited amount of information currently available on their structures. From a physiological point of view, a fruit results from the development of the tissue that supports the ovule of a plant. It is certain that tissue softening during the process of fruit ripening is related to breakdown of the organized structure of primary cell wall. The polygalacturonase-catalyzed degradation of pectic rhamnogalacturonan resulting in the dissolution of the middle lamella is probably the major contributor to tissue softening during fruit ripening.

Publisher Summary Plant galactomannans are reserve polysaccharides composed of linear chains of (1→4)-linked β-D-mannopyranosyl residues having single stubs of α-D-galactopyranosyl groups joined by (1→6)-linkages along the chain. These polysaccharides are widely used in industry especially in food, pharmaceuticals, cosmetics, paper products, paints and plasters, well-drilling and mining, and explosives and fire-fighting. The distribution of galactomannans in the plant kingdom is limited. The rich sources are the members of the family Leguminoseae. In context to the biosynthesis of plant galactomannans, it is concluded that D-galactose is stored in the seeds in the form of galactomannan, by random attachment of D-galactosyl groups to a main-chain of D-mannan. Seed galactomannans exhibits double physiological function. Firstly, they retain water by solvation and thereby prevent complete drying of the seeds, which can cause protein denaturation— in particular—the denaturation of those enzymes essential for seed germination. Secondly, the galactomannans serve as food reserves for the germinating seeds.

Galactokinase (EC 2.7.1.6) from the dormant seeds of Vicia faba was purified approximately 1300-fold with an 18% recovery through an eight-step procedure. The preparation showed the presence of only minor contaminations as judged by disc-gel electrophoresis. The native enzyme displayed a molecular weight of approximately 60 000 (determined by Sephadex G-100 gel-filtration) and the subunit value was 30 000. The isoelectric point of the enzyme was 5.3 and the amino acid analysis showed high percentage of acidic amino acids. The pH optimum of the enzyme was 7.3 at 25 degrees C. The relative activity for phosphorylating various monosaccharides followed the order, D-galactose greater than 2-deoxy-D-galactose greater than D-galactosamine; D-fucose, L-arabinose, L-galactose and D-glucose were not phosphorylated. Whereas ATP acted as an efficient phosphate donor, ADP, GTP and UTP were unable to act in this capacity. The Km and the V values of the substrates were determined. The metal ion requirement for the enzymic activity followed the order, Mg2+ greater than Co2+ greater than Mn2+ greater than Ni2+ greater than Ca2+. The enzymic reaction was inhibited by heavy metal ions and sulphydryl reagents indicating the participation of -SH group(s) in enzymic catalysis. Product inhibition was observed; galactose 1-phosphate and ADP were competitive and non-competitive inhibitors, respectively. Seed germination showed an increase in galactokinase level up to 24 h followed by a rapid decrease. The level of raffinose and stachyose decreased continually. The galactokinase level was found to be sufficiently high to phosphorylate the liberated galactose. No free galactose was observed at any stage of germination.

Publisher Summary This chapter discusses the role of lipid-linked sugars as intermediates in the biosynthesis of complex carbohydrates in plants. The addition of the activated glycosyl units to the nonreducing end of an acceptor is one of the general mechanisms involved in the polymerization of saccharides. The acceptor molecule is an oligomer or polymer whose nonreducing ends are the sites where new glycosyl groups are added. The in nitro synthesis of dolichyl phosphate was achieved for the first time with plant systems—namely, from isopentenyl diphosphate or mevalonate. The transfer of the oligosaccharide to protein is followed in vivo by the excision of the d -glucosyl residues; the nine d -mannosyl residues present in the transferred oligosaccharide probably remain in the inner core of the d -mannan. The general scheme for the glycosylation of N-glycosylically linked glycoproteins seems to be similar to that found in animal systems and yeast. The formation of practically all of the lipid-linked mono- and oligosaccharides present in animal tissues has also been obtained by using plant enzymes. However, in only a few cases the nature of the glycoprotein under consideration has been established.

1. The specificity of purified sweet-almond alpha-galactosidase has been investigated with 17 substrates. 2. Some of them exhibited inhibition at high substrate concentrations but others did not. Both substrate types were bound and hydrolysed at the same site on the enzyme. 3. The enzyme is specific for alpha-d-galactosides and beta-l-arabinosides. It did not hydrolyse beta-d-galactosides or alpha-d-glucosides. 4. Among galactosides the order of decreasing rates of enzymic hydrolysis was: aryl alpha-galactosides; sugars; alkyl alpha-galactosides. 5. All substituents in the aryl moiety of aryl alpha-galactosides enhanced V(max.), the electron-releasing (-sigma) groups being more effective than the electron-withdrawing (+sigma) groups. The substituent groups did not alter K(m) appreciably. 6. Implications of these results are discussed from a mechanistic viewpoint.

Background: We have previously reported an increase in symptoms of anxiety in patients with posttraumatic stress disorder (PTSD) following administration of the β2-antagonist yohimbine, which stimulates brain norepinephrine release. Preclinical studies show decreased metabolism in the neocortex and the caudate nucleus with high-dose yohimbine-induced norepinephrine release, but low levels of norepinephrine release result in an increase in metabolism in these areas. Methods: We used positron emission tomography and fludeoxyglucose F 18 to measure brain metabolism in Vietnam combat veterans with PTSD (n=10) and healthy age-matched control subjects (n=10), following administration of yohimbine (0.4 mg/kg) or placebo in a randomized, double-blind fashion. Results: Yohimbine resulted in a significant increase in anxiety in the patients with PTSD, but not in healthy subjects. There was a significant difference in brain metabolic response to yohimbine in patients with PTSD compared with healthy subjects in prefrontal, temporal, parietal, and orbitofrontal cortexes. Metabolism tended to decrease in patients with PTSD and increase in healthy subjects following administration of yohimbine. Conclusion: These findings are consistent with our previous hypothesis of enhanced norepinephrine release in the brain with yohimbine in patients with PTSD.

Fruit ripening is associated with cell wall modifications. The present review focuses on cell wall components and the nature of noncovalent and covalent interactions in the primary cell wall. The role of structural protein cross-links are evaluated within the context of cell wall-mediated changes in texture during fruit ripening. The article discusses molecular approaches in fruit cell wall interactions to regulate processes in fruit ripening in order to improve post-harvest textural characteristics.

Liver fats ofCarcharias limbatus andPristis cuspidatus from the West Coast of India have been studied. Lithium salt-acetone and lead salt-alcohol methods were adopted for the resolution of the fatty acids. An efficient, electrically heated and packed column has been employed for fractionation of the methyl esters of fatty acids. These fats have been found to have a high content of saturated fatty acids and thus belong to the fourth group of Tsujimoto's classification of Elasmobranch fish liver fats. Analyses of the liver oils from the same species of fish show that there is a tendeney towards saturation or hydrogenation of polyethylenic acids accompanied by an increasing concentration of unsaponifiable matter, which is mainly alcohol ethers of the selachyl type, in accordance with Lovern's views.