Identification of a peptide sequence involved in association of subunits of yeast hexokinases

The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.     

Chemistry and subunit structure of yeast hexokinase isoenzymes

Evidence from ultracentrifugation, sodium dodecyl sulfate electrophoresis, peptide mapping, and carboxypeptidase A digestion allows the conclusion that the two native hexokinases, P-I and P-II, consist of polypeptide chains having molecular weights slightly higher than 50,000. It was demonstrated that some preparations are contaminated with a protease, and that this impurity caused erroneous results in sodium dodecyl sulfate electrophoresis and carboxypeptidase A digestion.Amino acid analyses indicated that both P-I and P-II contain four cysteine, four tryptophan, and eleven methionine residues per mole. In contrast, P-I contains eight, and P-II five, histidine residues per mole. Many of the differences in amino acid composition are small, but reproducible.Peptide mapping indicated that many segments of P-I and P-II have identical sequences. There were about 27 common tryptic peptides, and about 16–19 unique to each form. In addition, both isozymes were found to have the same amino terminus, valine, and the same carboxy terminus, alanine; some evidence for a difference in the penultimate residue at the carboxy terminus was indicated.     

Catalytic activity with associated and dissociated forms of the yeast hexokinases.

In agreement with previous glucose binding data, kinetic studies of the yeast hexokinases at high protein concentration show that the dimeric forms P-I and P-II require much higher glucose concentrations for half-maximal rates than do the monomeric forms S-I and S-II, but each P form reaches the same V value as the corresponding S form. Raising the temperature from 5 °C to 24 °C causes an apparent dissociation of the P forms, most pronounced in the case of P-I, as evidenced by a lowering of glucose K_M values toward those of the S forms. Similarly, kinetic measurements at low protein concentrations, where the P forms are presumably fully dissociated, give identical glucose K_M values for the P and S forms.     

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.     

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.     

The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini.

cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa

Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the North-Western Himalayas. Extensive harvesting for pharmaceutical purposes, lack of organized cultivation and unorganized methods of uprooting the plants because of unawareness has brought an endangered status to this important herb in nature. The medicinal property of this plant is attributed to monoterpenoid picrosides. The influence of developmental status of different growth stages on picrosides content is poorly understood in Picrorhiza kurroa. Picroside-I (P-I) content increased from 0.05 % to 0.76 % in different growth stages of shoots. Significant increase in the contents of P-I (0.15–0.50 %) and Picroside-II (P-II) (0.1–0.45 %) was observed in rhizomes of different developmental stages. Highest amounts of P-I (8.7 %) and P-II (5.3 %) was detected in uppermost part of mature dried rhizomes compared to bottom part with 2.9 % and 2.2 % of P-I and P-II, respectively. P. kurroa grown at high altitude (Sairopa, 4,500 amsl) showed 1.75-folds increase in P-I in leaves whereas exponential increase in the P-I content was detected (0.05–1.7 %) in the leaves of different developmental stages (L1-L5) of P. kurroa grown at lower altitude (Jagatsukh, 1,900 m). Variable amounts of P-I and P-II in different growth and developmental stages of P. kurroa imply importance of selection of plant material (rhizomes and roots). The study undertaken explored the status of metabolites accumulation and biosynthesis in the field grown plants of P. kurroa where not only environmental parameters but different morphogenetic stages of its developmental cycles, different age groups and different parts of plantlets were extensively analysed and estimated for medicinally important picrosides.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.     

Biochemical biomarkers in gills of mangrove oyster Crassostrea rhizophorae from three Brazilian estuaries

Responses of biochemical biomarkers were evaluated in gills of immature adult mangrove oyster Crassostrea rhizophorae collected in three estuarine regions along the Brazilian coast. In each region, ten oysters were collected in one reference site (R) located far from pollution sources, and in two polluted sites (P-I and P-II sites) located in another water body with similar characteristics. P-I site is located close to recognized pollution sources while P-II site is in the same water body, but far from pollution sources. At the Paranaguá Bay (Southern Brazil), polluted sites receive domestic, harbor and phosphate fertilizer plant discharges. High lipid peroxides (LPO) content was observed in winter oysters from the P-I site. In summer, higher catalase activity was observed in these oysters. In the Piraquê region (Southeastern Brazil), polluted sites receive domestic and agricultural effluents. Lower total oxyradical scavenging capacity (TOSC) towards peroxyradicals was observed in summer oysters from both P-I and P-II sites. In the Itamaracá region (Northeastern Brazil), polluted sites receive paper mill and caustic soda and chlorine factories effluents. Increased glutathione S-transferase (GST) activity was observed in oysters from the P-I site in both summer and winter. At Paranaguá Bay (higher latitude), no seasonal differences were observed in oysters from the R site, suggesting that temperature was not an important factor influencing biomarkers levels. Lower GST activity was observed in oysters from the R site of the Itamaracá Bay (lower latitude) in winter and summer. Taken together, data obtained point to responses of biomarkers in oysters from polluted sites of the three estuarine regions analyzed, indicating the need for future monitoring of the biological effects of contaminants in these environments. They also point to the relevance to consider both season and latitude as factors influencing biomarker responses in environmental contamination monitoring programs.     

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation

Picrorhiza kurroa is a medicinally important, high altitude perennial herb, endemic to the Himalayas. It possesses strong hepato-protective bioactivity that is contributed by two iridoid picroside compounds viz Picroside-I (P-I) and Picroside-II (P-II). Commercially, many P. kurroa based hepato-stimulatory Ayurvedic drug brands that use different proportions of P-I and P-II are available in the market. To identify genetically heterozygous and high yielding genotypes for multiplication, sustained use and conservation, it is essential to assess genetic and phytochemical diversity and understand the population structure of P. kurroa. In the present study, isolation and HPLC based quantification of picrosides P-I and P-II and molecular DNA fingerprinting using RAPD, AFLP and ISSR markers have been undertaken in 124 and 91 genotypes, respectively. The analyzed samples were collected from 10 natural P. kurroa Himalayan populations spread across four states (Jammu & Kashmir, Sikkim, Uttarakhand and Himachal Pradesh) of India. Genotypes used in this study covered around 1000 km geographical area of the total Indian Himalayan habitat range of P. kurroa. Significant quantitative variation ranging from 0.01 per cent to 4.15% for P-I, and from 0.01% to 3.18% in P-II picroside was observed in the analyzed samples. Three molecular DNA markers, RAPD (22 primers), ISSR (15 primers) and AFLP (07 primer combinations) also revealed a high level of genetic variation. The percentage polymorphism and effective number of alleles for RAPD, ISSR and AFLP analysis varied from 83.5%, 80.6% and 72.1%; 1.5722, 1.5787 and 1.5665, respectively. Further, the rate of gene flow (Nm) between populations was moderate for RAPD (0.8434), and AFLP (0.9882) and comparatively higher for ISSR (1.6093). Fst values were observed to be 0.56, 0.33, and 0.51 for RAPD, ISSR and AFLP markers, respectively. These values suggest that most of the observed genetic variation resided within populations. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian based STRUCTURE grouped all the analyzed accessions into largely region-wise clusters and showed some inter-mixing between the populations, indicating the existence of distinct gene pools with limited gene flow/exchange. The present study has revealed a high level of genetic diversity in the analyzed populations. The analysis has resulted in identification of genetically diverse and high picrosides containing P. kurroa genotypes from Sainj, Dayara, Tungnath, Furkia, Parsuthach, Arampatri, Manvarsar, Kedarnath, Thangu and Temza in the Indian Himalayan region. The inferences generated in this study can be used to devise future resource management and conservation strategies in P. kurroa.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00972-w.     

Evidence for two α-amanitin-resistant RNA polymerases in vegetative amoebae of Dictyostelium discoideum

Three DNA-dependent RNA polymerases (EC 2.7.7.6), P-I, P-II and P-III, have been isolated from the sonicated nuclear extract of vegetative amoebae of $\text{Dictyostelium discoideum}$ by phosphocellulose chromatography. P-I was inhibited by α-amanitin, while P-II and P-III were not. Rifampicin did not prevent all the polymerase activities. These polymerases were more active in the presence of Mg²⁺ than Mn²⁺. P-III was reduced in the enzyme activity by being passed through DEAE-Sephadex column and not obtained from the nuclear extract of amoebae at the culmination stage during morphogenetic development.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Effect of calcium on the endogenous phosphorylation of mouse brain microsomes in vitro

1. The endogenous phosphorylation of mouse brain microsomes was studied using the technique of acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). 2. It was found that specific proteins and lipids in brain microsomes were phosphorylated by the terminal phosphate of ATP under appropriate conditions. Six peaks of radioactivity were observed on SDS-polyacrylamide gel electrophoresis of 32Pi-labelled brain microsomes. The peaks were designated as P-I, P-II, P-III, P-IV, P-V, and P-VI. The peaks from P-I to P-V, which consist of phosphoproteins, underwent rapid dephosphorylation. On the other hand, P-VI, which consists of phospholipids, remained unaffected even after the complete hydrolysis of added ATP. 3. With the addition of 100 muM CaCl2 to the assay medium, the phosphorylation of brain microsomal proteins was stimulated; in the regions of P-I, P-II, and P-III, the amounts of 32Pi incorporation were approximately twice the 32Pi incorporation in the absence of Ca2+. On the other hand, 32Pi incorporation into P-VI was unaffected irrespective of the presence or absence of 100 muM CaCl2. In the presence of higher concentrations of Ca2+ (1-10 mM), the phosphorylation of all components was inhibited.