STUDIES ON BLACK GRAM (Phaseolus mungo L.) TRYPSIN INHIBITOR

ABSTRACT A trypsin inhibitor isolated from black gram (Phaseolus mungo L.) had 75 amino acid residues with an estimated molecular weight of 7892. The kinetic constants K_m and V_max as evaluated by the Dixon and Cornish-Bowden plots were 2.7 × 10^?5and 6 × 10^?3M/min, respectively. The dissociation constants of the enzyme-inhibitor complex (Ki) and the enzyme-inhibitor-substrate complex (Ki') were respectively 4 × 10^?7M and 1.9 × 10^?6M. Trypsin inhibition by black gram trypsin inhibitor was of a linear-mixed type. Chemical modification studies suggested the possible involvement of lysine and arginine at the active site of the inhibitor.     

Inhibition of cholesterol biosynthesis and acetyl-coenzyme A synthetase by bovine milk and orotic acid.

Pasteurized, homogenized bovine milk or orotic acid in solution at final concentrations varying from 3.3 μM to 322 μM in rat liver homogenates inhibited the incorporation of [1-Carbon 14] acetate but not [1-Carbon 14] acetyl-coenzyme A, 3-hydroxy-3-methyj-[Carbon 14] glutaryl-coenzyme A or [5-hydrogen 3] mevalonic acid into cholesterol. Milk inhibited cholesterol biosynthesis up to 72 ± 10% before acetyl-coenzyme A formation, thus indicating that acetyl-coenzyme A synthetase is the affected enzyme. Kinetics of the inhibition were studied with acetyl-coenzyme A synthetase purified from yeast. From a Line-weaver-Burk plot of the inhibition of yeast acetyl-coenzyme A synthetase, orotic acid is a noncompetitive inhibitor of acetyl-coenzyme A synthetase. A Michaelis constant was 6.0 × 10^?4 M for acetate with the yeast enzyme while a K_i was 6.6 × 10^?5 M for orotic acid. The approximate point of 50% inhibition with rat liver enzyme was 7 × 10^?6 M orotic acid indicating the mammalian enzyme may be more sensitive to the orotic acid. Nicotinic acid also inhibited the yeast enzyme. Fifty percent inhibition required a relatively high final concentration–about 12 mM. Neither raw milk nor pasteurized, homogenized milk inhibited 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase that had been partially purified from rat liver.     

A Fluorescence‐Based Method for Cyanate Analysis in Ethanol/Water Media: Correlation between Cyanate Presence and Ethyl Carbamate Formation in Sugar Cane Spirit

Based on the fluorescence properties of 2,4‐(1H,3H)‐quinazolinedione, a product of the reaction between cyanate and 2‐aminobenzoic acid, a simple, sensitive, selective, and reproducible method for the cyanate analysis in aqueous ethanolic media is proposed. In this method, λ_exc and λ_em are 310 and 410 nm, respectively, and the limits of detection and quantification are 2.2 × 10^?7 and 6.7 × 10^?7 mol/L, respectively. Under optimal conditions (pH = 4.5, 40% ethanol), a concentration of 5.0 × 10^?6 mol/L cyanate can be determined in a single measurement, at a 95% level of confidence, with an uncertainty of ± 0.13 × 10^?6 mol/L. Cyanide, thiocyanate, chloride, nitrate, and sulfate ions, as well as urea and urethane in concentrations 1 × 10³ higher than that of cyanate do not interfere with the measurement. The methodology was applied to cyanate analyses in the different fractions of the sugarcane distillate and the data strongly suggest a correlation between the presence of urea in wine, and the cyanate and ethyl carbamate concentrations in the spirit.     

Myrosinase activity of cruciferous vegetables

Myrosinase activity in partially purified extracts of 12 cruciferous vegetables and an acetone powder preparation of Sinapis alba L. (white mustard) was determined by the initial rate of glucose formation from glucosinolate hydrolysis using a coupled assay. Of the species studied Raphanus sativus L. (radish, 12.8±0.7 μmol min^?1g^?1 powdered tissue) had the greatest myrosinase activity, and Brassica campestris L. ssp. rapifera (turnip) and Nasturtium officinalis R.Br. (watercress) (0.6±0.1 and 0.8±0.03 μmol min^?1g^?1 powdered tissue respectively) the least. The sub-species of Brassica oleracea studied all had similar myrosinase activity (ca 2.5±0.2μmolmin^?1g^?1 powdered tissue) except B. oleracea L. var. gemmifera D.C. (Brussels sprouts) and B. oleracea L. var. capitata L. (white cabbage) which had higher activities (7.6±0.1 and 5.2±0.2μmol min^?1g^?1 powdered tissue respectively). The effect of ascorbate concentration upon the myrosinase activity of six of the crucifers studied and the white mustard preparation, revealed that the ascorbate concentration necessary to promote maximal activity varied with species. A concentration of 0.9mM ascorbate maximally activated radish and turnip myrosinase, while red cabbage, watercress, white mustard and Brussels sprouts were maximally activated at 2.0, 3.0, 5.0 and 0.7–1.0mM ascorbate respectively. Two peaks of maximal myrosinase activity, occurring between 0.9 and 1.0mM and at 3.0mM ascorbate, were found for B. oleracea L. var. botrytis L. subvar. cauliflora D.C. (cauliflower).     

Occurrence of ochratoxin A,fumonisin B2 and black aspergilli in raisins from Western Greece regions in relation to environmental and geographical factors

The presence of ochratoxin A (OTA), fumonisin B₂ (FB₂) and black aspergilli in raisins from Western Greece regions (Messinia, Corinthia, Achaia, Ilia and Zante Island) was investigated in relation to the different geographic and climatic conditions in the 2011 growing season. The biseriate species Aspergillus niger “aggregate” and A. carbonarius were mainly identified. The population of A. niger “aggregate” species occurred in all raisin samples at colony-forming units (CFU) concentrations significantly higher (mean 2.2 × 10⁵ CFU g^?1 homogenate) than those of A. carbonarius population (mean 4.9 × 10³ CFU g^?1 homogenate), which occurred in 80% of the raisin samples. OTA was found in 73% of the samples at levels ranging from 0.1 µg kg^?1 to 98.2 µg kg^?1, with the highest level occurring in a raisin sample from Ilia that also contained the highest level of A. carbonarius. The European Union legal limit for OTA was exceeded in 15% of the raisin samples. FB₂ was found in 29% of the raisin samples at levels ranging from 7.1 µg kg^?1 to 25.5 µg kg^?1, with 20% of the samples co-occurring with OTA. Principal-component analysis was applied to levels of mycotoxins, fungal contamination, geographical data and environmental conditions recorded in the harvesting (August) or drying (September) period. Principal-component analysis clearly indicated a good direct correlation of rainfall and relative humidity with OTA and A. carbonarius contamination. A lack of clustering was observed when A. niger and FB₂ contamination were considered. This is the first report on the co-occurrence of the mycotoxins OTA and FB₂ in dried vine fruits from Greece.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

Biochemical and morphological characterization of a new fungal contaminant in balsamic and cider vinegars

In this study we have determined the morphological, some physiological and biochemical characteristics of a fungal strain isolated from balsamic and cider vinegars. The examination of fungal contamination of 100 balsamic and cider vinegar samples showed that 90% contained more than 10⁴ colony forming units (CFU ml^?1), but in most samples (65%) values of more than 10⁵ CFU ml^?1 were obtained. The most frequent contamination was with Monascus sp. (80% of the samples examined showed 2 × 10³ to 6 × 10⁵ CFU ml^?1). This mould can produce secondary metabolites such as citrinin and monacolin K_L (lactone form), and monacolin K_A (acid form). Using high-performance liquid chromatography (HPLC) with fluorescence detection, citrinin was found in 68% of the samples. The concentration of these mycotoxins varied between 1.6 × 10^?2 µg ml^?1 and 7.2 × 10^?2 µg ml^?1. The concentrations of citrinin were very low, and we can anticipate that this compound at these concentrations has no toxic effects on renal cells. Monacolin was not detected in any sample studied.     

PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE ISOZYMES FROM CANOLA (Brassica napus cv, WESTAR) SEED

Four isozymes, I', II'a, II'b and III’ of lipoxygenase (EC 1.13.11.12) from Canola (Brassica napus, cv Westar) seed were purified by successive chromatography on ion-exchange and size-exclusion columns using a Fast Protein Liquid Chromatograph (FPLC). The homogeneity of each isozyme was demonstrated by a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weights of isozymes I', II'a, II'b and III’ were 72, 000, 106, 000, 78, 000 and 62, 000, respectively. The optimum pH for lipoxygenase activity was 6.5 for isozyme I’ and 6.0 for isozymes II'b, II'b and III'. Apparent K_m value for isozymes I', II'a, II'b and III’ were 5.5 × 10^?4 M, 3.4 × 10^?4 M, 4.0 × 10^?4 M and 3.8 × 10^?4 M, respectively. Isozyme I’ displayed preferential activity towards monolinoleate and dilinoleate, while isozyme II'a demonstrated preferential activity towards dilinoleate followed by mono- and trilinoleate. No enzymatic activity was observed with both isozymes I’ and II'a toward free linoleic acid. Isozyme II'b showed activity towards free linoleate as well as mono-, di- and trilinoleate. Isozyme III’ showed preferential activity towards free linoleate. The activity of isozymes I’ and II'a was inhibited completely by the addition of 10 mM and 4 mM KCN, respectively, while the addition of 3 mM and 10 mM KCN to isozymes II'b and III', respectively, increased activity by approximately 20%.     

Purification and Biochemical Characterization of Peroxidase Isolated from White Cabbage (Brassica Oleracea var. capitata f. alba)

Peroxidase enzyme was purified for the first time from white cabbage (Brassica oleracea var. capitata f. alba) in a single step using affinity chromatography and some biochemical characteristics of the purified enzyme were determined. The peroxidase was purified 24.7-fold with an overall recovery of 4.3% and a specific activity of 964.5. The molecular weight of the purified peroxidase was approximately 73.2 kDa as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it showed maximum activity at pH 6.5 and 30°C. For the guaiacol substrate, the K_M and V_max values were found as 3.19 mM and 0.2 EU/mL, respectively. Additionally, the IC₅₀ and K_i values were determined as 0.517 and 0.994 ± 0.453 mM, respectively, for 4-aminobenzohydrazide. 4-amino benzohydrazide showed non-competitive inhibition.     

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

Inhibition of lipase from rice (Oryza sativa) by diethyl-p-nitrophenyl phosphate

The inhibition of rice bran lipase (RBL) by diethyl-p-nitrophenyl phosphate was studied with reference to kinetics, nature of inhibition and also elucidate the effect of the inhibitor on the structure—function of the enzyme. Enzyme activity measurements shows that the inhibitor is more effective at 0.050 mM concentration of diethyl-p-nitrophenyl phosphate and the activity is 50% at this level of inhibitor concentration. The affinity of substrate for the enzyme was observed by the increase in the velocity of the reaction with increase in the substrate concentrations and double reciprocal plot indicates that the inhibition followed a competitive in nature and inhibition constant K _i is found to be 0.016 mM at pH 7.0. The decrease in apparent thermal denaturation temperature to 4 °C compared to control indicates the destabilization of enzyme in the presence of inhibitor. Fluorescence spectral measurements suggests that pronounced quenching of fluorescence intensity of RBL occurs at higher concentrations of diethyl-p-nitrophenyl phosphate and ‘K _a’ value was found to be 2.4 × 10⁴ M⁻¹ with free energy change ΔG^o—26 kJ/mol at 30 °C suggesting strong binding between the enzyme and the inhibitor with microenvironmental changes occur at the active site or in the neighbourhood of active site. The far UV-CD data suggest that there is no significant changes in the conformation of the enzyme as a result of binding of diethyl-p-nitrophenyl phosphate. These results indicate that diethyl-p-nitrophenyl phosphate is a inhibitor of RBL and binds to the enzyme in brining about inhibition without any structural alterations.     

Contamination of food samples from Malaysia with polychlorinated dibenzo-p-dioxins and dibenzofurans and estimation of human intake

A total of 126 food samples, categorised into three groups (seafood and seafood products, meat and meat products, as well as milk and dairy products) from Malaysia were analysed for polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The concentration of PCDD/Fs that ranged from 0.16 to 0.25 pg WHO₀₅-TEQ g^?1 fw was found in these samples. According to the food consumption data from the Global Environment Monitoring System (GEMS) of the World Health Organization (WHO), the dietary exposures to PCDD/F from seafood and seafood products, meat and meat products, as well as milk and dairy products for the general population in Malaysia were 0.064, 0.183 and 0.736 pg WHO₀₅-TEQ kg^?1 bw day^?1, respectively. However, the exposure was higher in seafood and seafood products (0.415 pg WHO₀₅-TEQ kg^?1 bw day^?1) and meat and meat products (0.317 pg WHO₀₅-TEQ kg^?1 bw day^?1) when the data were estimated using the Malaysian food consumption statistics. The lower exposure was observed in dairy products with an estimation of 0.365 pg WHO₀₅-TEQ kg^?1 bw day^?1. Overall, these dietary exposure estimates were much lower than the tolerable daily intake (TDI) as recommended by WHO. Thus, it is suggested that the dietary exposure to PCDD/F does not represent a risk for human health in Malaysia.     

A Biomimetic Sensor with Signal Enhancement of Ferriferrous Oxide-Reduced Graphene Oxide Nanocomposites for Ultratrace Levels Quantification of Methamidophos or Omethoate in Vegetables

In this study, we designed and synthesized Fe₃O₄/reduced graphene oxide (Fe₃O₄/rGO) and MIL@MIP, which were characterized using transmission electron microscopy (TEM), X-ray diffraction, Fourier transform infrared (FT-IR), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). Here, reports a novel biomimetic electrochemical sensor (BECS) sensitized with Fe₃O₄/reduced graphene oxide (Fe₃O₄/rGO) nanocomposite using MIL@MIP film as recognition element for ultrasensitive quantification of methamidophos or omethoate. The BECS offered fast responses for methamidophos and omethoate with wide linear response ranges of 1.0 × 10^?7–1.0 × 10^?12 and 1.0 × 10^?7–1.0 × 10^?13 mol/L, respectively. Under optimized conditions, the limit of detection (LOD; S/N = 3) of the BECS method was 2.67 × 10^?13 mol/L for methamidophos and 2.05 × 10^?14 mol/L for omethoate. The BECS was applied to detection of methamidophos and omethoate in spiked cucumber and kidney bean samples, and good recoveries varied from 88.2 to 96.9 %. This proposed method was adopted for the determination of the methamidophos or omethoate residues in the rape sample, and the results were also verified using the method of gas chromatography.     

Neutral and acidic ascorbate oxidases from satsuma mandarin (Citrus unshiu Marc): Isolation and properties

The neutral and acidic forms of ascorbate oxidase (AAO) were isolated and purified from young fruits of satsuma mandarin by a combination of (NH₄)₂SO₄ fractionation, ion-exchange and hydrophobic chromatography, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respectively. Neutral AAO was stable in the range of pH 5 and 8 and exhibited optimal activity at pH 5.5 and 45°C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5.5 and 50°C. When L-ascorbate and D-isoascorbate were used as substrates, K_m values of neutral AAO were 2.98 × 10^?4 M and 3.36 × 10^?4 M and those of acidic AAO were 1.99 × 10^?4 M and 1.86 × 10^?3 M, respectively. The activities of the two forms of AAO were totally inhibited by metabisulphite and cyanide, substantially suppressed by higher concentrations of diethyldithiocarbamate and fluoride, and not inhibited at all by monovalent and divalent metal ions tested.     

REGULATION OF PHENYLALANINE AMMONIA-LYASE ENZYME IN ANNONA FRUIT: KINETIC CHARACTERISTICS AND INHIBITORY EFFECT OF AMMONIA

In this work, we analyzed the kinetic properties of phenylalanine ammonia‐lyase (PAL) extracted from “cherimoya” (Annona cherimola Mill.) fruits ripened at ambient temperature (20C) and stored under several environmental conditions, including high CO₂ levels (20%) and low temperature (6C). The effect of different ammonia‐related compounds on cherimoya PAL activity was also evaluated. PAL exhibited two different K_mvalues for L‐phenylalanine (L‐Phe ) and negative substrate cooperativity, with Hill coefficient (n_app) values reaching 0.64 and 0.71 for low temperature and high CO₂ levels, respectively. The kinetic analysis revealed that ammonia produced mixed inhibition of PAL enzyme, with inhibition constants (K_i and K_i′) values of 0.57 ± 0.2 mM and 2.54 ± 0.2 mM. We propose that the regulation of PAL by ammonia inhibition and the negative cooperativity may be essential in adjusting the active phenylpropanoid metabolism in Annonas to the requirement of L‐Phe and in consequence, to the carbon skeleton demand for other anabolic pathways.     

Spectrofluorometric Method for Determination of Al3+ with 3-Hydroxy-2-Naphthalenecarboxylic Acid and its Application to Milk Samples

On the basis of affinity of Al³⁺ ions toward functional groups of 3-hydroxy-2-naphthalenecarboxylic acid (3H2NA), a high stable complex between Al³⁺ and 3H2NA in 5 % methanol/water solution was established. The mole ratio method showed that a stable complex by taking part of acetate and 3H2NA with the stoichiometric ratio of 1:1:1 and stability constant of 8.6?×?10⁶ is formed. Under the optimum conditions for the complex formation, a rapid spectrofluorometric determination of trace amounts of Al³⁺ (λ _ex?=?366 nm, λ _em?=?460 nm) was proposed. The optimum conditions involve 3H2NA concentration of 1.0?×?10^?5 M, buffering pH of 4.5 (acetic/acetate system), and ionic strength of 0.01 M. The fluorescence intensity exhibited a good linearity against the Al³⁺ concentration in the range 5.0?×?10^?8–5.0?×?10^?6 M with the detection limit of 1.3?×?10^?8 M. No considerable interference was observed due to the presence of coexisting anions and cations. The method was applied successfully to the determination of trace amounts of Al³⁺ in milk. Validation of the measurements was confirmed using graphite furnace atomic absorption spectrophotometry.     

Factors influencing permeability of the cell membrane of the osmotolerant yeast Saccharomyces rouxii grown in the presence and absence of 18% NaCl. I. Na+/K+-activated Mg2+-dependent ATPase

It was found that cells of Saccharomyces rouxii contain an ouabain-inhibited ATPase, assumed to be an Na⁺/K⁺-activated Mg²⁺-dependent ATPase, which could serve as a sodium pump protecting the cells in a high salt environment. Twenty-two cell homogenates or supernatants (centrifuged at 3000 × g) grown without added salt in the medium contained sufficient total ATPase activity to liberate (on average) 0.225 μM P_i min^?1 mg^?1 protein. The percentage of total ATPase inhibited by the addition of ouabain (1 × 10^?4 M) varied from 7 to 100%. Cell homogenates or supernatants from cells grown in the presence of 18% NaCl in the media contained sufficient ATPase activity to liberate (on average) 0.114 μM P_i min^?1 mg^?1 protein, about 50% of the total ATPase activity found in the non-salt grown cells. The percentage of total ATPase activity inhibited by ouabain ranged from 16 to 100%. Although the non-salt-grown cells contained approximately double the total ATPase activity of the salt-grown cells. there was evidence that the percentage of total ATPase that is ouabain sensitive (Na⁺/K⁺-activated ATPase) is higher in the salt-grown cells. Also, cells of S. rouxii grown in media without added NaCl, recovered by centrifugation and transferred to media containing 18% NaCl for 16 h and again recovered by centrifugation, homogenized and centrifuged at 10 000 × g contained 61.2% ouabain-sensitive ATPase compared with 21.3% ouabain-sensitive ATPase in the cells before adaptation to the high salt environment.     

Amorphophallus paeoniifolius corm: A potential source of peroxidase for wide applications

Peroxidases have wide applications in different areas including chemical synthesis, medicine, food industry, bioremediation of wastewater, biosensing, and biotechnology. Amorphophallus paeoniifolius (elephant foot yam) is an attractive source of enzymes; however, no effort has been made to assess peroxidase enzyme from this source. Therefore, the objectives of this study were to evaluate and compare peroxidases from corms of elephant foot yam and roots of Dacus carota (carrot) and Aramoracia rusticana (horseradish). Elephant foot yam corm peroxidase (ECP) demonstrated 4.5 times higher specific activity compared with carrot root peroxidase (CRP). ECP showed retention of high activity over a broad pH range and had higher temperature optima and thermal stability compared with CRP and horseradish peroxidase (HRP). The calculated K_M values of ECP, CRP, and HRP for the substrates guaiacol and hydrogen peroxide were 4.5 mM and 307.8 µM, 4.6 mM and 55.5 µM, and 3.5 mM and 413.0 µM, respectively. Peroxidases are used for the bioremediation of wastewater contaminated with hazardous aromatic compounds. Heavy metals such as cadmium (Cd²⁺), lead (Pb²⁺), and toxic compounds such as sodium azide (NaN₃) are often present in wastewater along with aromatic compounds. Results showed activation of ECP and inhibition of HRP at 250 and 500 µM concentrations of Cd²⁺ and Pb²⁺. Treatment with 1 mM NaN₃ led to 8.4%, 55.5%, and 11.0% inhibition in the activities of ECP, CRP, and HRP, respectively. Overall, our results suggest that elephant foot yam can be used as a rich and convenient source of peroxidase for various applications including wastewater remediation.     

Biochemical similarity of Schizosaccharomyces pombe ras1 protein with RAS2 protein of Saccharomyces cerevisiae

Schizosaccharomyces pombe contains single ras oncogene homologue, ras1, that functions in the signal transduction pathway conducting the cell's mating processes. To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein from Saccharomyces cerevisiae and mammalian ras proteins. The purified ras1 protein showed a remarkably high K_d value for GDP binding (178 nM) and for binding with ATP. In contrast, the K_d value for GTP binding and the rate of GTPase activity were 64 nM and 77 × 10^?6 s^?1 at 37°C, respectively; both were higher than normal p21^ras protein, but at the same level as the RAS2 protein. We directly measured rate of GTP binding and GDP binding which were 3.9 × 10^?3 s^?1 and 1.8 × 10^?3 s^?1 at 30°C, respectively. On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several-fold lower than the binding rate. These results suggest that the release of the guanine nucleotide is the rate-limiting step in the ras–GTP/GDP cycle. As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transduction pathway in the cells.     

THERMAL PROPERTIES OF RADISH AND ALFALFA SEEDS

Thermal conductivity, specific heat, and density were measured in a temperature range of 30C to 80C, and moisture content of 4.6% to 6.5% for radish seeds, 5.0% to 7.3% for alfalfa seeds typically encountered in the radio frequency dielectric heating for seeds. Thermal conductivity and specific heat were determined using a linear heat source probe and a modified calorimeter method, respectively. Thermal conductivity of radish and alfalfa seeds ranged from 0.050 to 0.091 W/mC (SD: 0.011) and 0.075 to 0.120 W/mC (SD: 0.012), respectively. Specific heat was in the range of 0.290 to 0.479 kJ/kgC (SD: 0.051) for radish seeds and 0.333 to 0.593 kJ/kgC (SD: 0.076) for alfalfa seeds. Density of radish and alfalfa seeds ranged from 684 to 675 kg/m³ (SD: 5.592) and 819 to 804 kg/m³ (SD: 8.921), respectively. Thermal diffusivity of the seeds was also determined, and the values varied from 25.13 × 10^?8 to 34.47 × 10^?8 m²/s for radish seeds and 27.14 × 10^?8 to 29.73 × 10^?8 m²/s for alfalfa seeds. Regression models were fitted to the experimental data for each thermal property, and showed second order polynomial relationships with temperature and moisture content.