Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Four isozymes of β-N-acetylhexosaminidase (β-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated β-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-β-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. β-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to β-NAHAs from other plant sources.     

Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans

Storage protein hydrolysis in the cotyledons of germinating mung beans (Phaseolus aureus Roxb.) was examined by histochemical techniques, and the autolytic capacity of isolated protein bodies was studied with biochemical methods. The localization of endopeptidase activity within the cotyledons was studied using an India ink-gelatin film technique. After 24 hours of imbibition, a low level of endopeptidase activity was found throughout the storage tissues of the cotyledons. A marked increase in activity was noted in cells farthest from the vascular bundles 48 to 60 hours after the start of imbibition. The decrease in storage protein followed the same spatial distribution starting in the cells farthest from the bundles. The cotyledons contain a population of cells in various stages of endopeptidase activity enhancement and storage protein degradation. A wave of endopeptidase activity moves progressively through the cotyledons towards the vascular bundles leaving behind areas devoid of stored reserves and low in endopeptidase activity. Observations on the morphology of protein bodies during germination indicate that the membrane surrounding them remains intact, while the reserves disappear. This result suggests that the protein bodies may be undergoing autolysis. To determine whether this may indeed be the case, protein bodies were isolated from the meal of mung bean seeds using an aqueous medium containing 80% glycerol. The protein body preparations and the cytoplasm were assayed for the presence of a number of enzymes which may be involved in the breakdown of the storage proteins. The protein bodies contained all, or nearly all, of the carboxypeptidase, α-mannosidase, N-acetyl-β-glucosaminidase, and caseolytic activity. The cytoplasm contained all, or most, of the leucine aminopeptidase and the trypsin-like activity (benzoyl arginine-p-nitroanalide as substrate). Incubation of the isolated protein bodies resulted in the release of amino acids. An analysis of the products of hydrolysis indicated that very little, if any, storage protein was being hydrolyzed during the incubation. Hydrolysis of the storage proteins present in the protein bodies was greatly accelerated by the addition of extracts from the cotyledons of 4-day-old seedlings. The results suggest that new enzymic activities not present in the protein bodies isolated from dry seeds must either be activated or synthesized and possibly added to the protein bodies before storage protein breakdown can begin.     

Isolation and Characterization of Protein Bodies in Lupinus angustifolius

Using Nycodenz, a novel density gradient medium, we isolated intact protein bodies from developing seeds of Lupinus angustifolius L. (cultivar Unicrop) and achieved excellent separation from the endoplasmic reticulum, mitochondria, and other organelles. The distribution of the storage protein conglutin-β was taken as evidence that up to 96% of the protein bodies remained intact on the gradients and banded at 1.25 grams per milliliter. The protein bodies also contained the three other abundant proteins present in L. angustifolius seeds: conglutins-α, -γ, and -δ. Pulse labeling experiments were carried out to determine the site of proteolytic processing of conglutin-α, a legumin-like 11Svedberg unit storage protein. Cotyledons aged either 33 or 40 days after flowering were pulsed with [³H]leucine. Protein bodies obtained from the cotyledons aged 33 days after flowering contained only the labeled precursors of conglutin-α with molecular weights 85,000, 72,000, and 64,000, even after a 4 hour chase of the radioactivity. Protein bodies obtained from the cotyledons aged 40 days after flowering contained the same radioactive precursors if the tissue had been pulsed for 2 hours, and the processing products of these precursors when the tissue had been chased for 4 hours. These studies confirm that the subcellular location of proteolytic cleavage of this legumin-like protein is the protein body, that this activity is detected only in protein bodies from lupin seeds aged between 33 and 40 days of seed development after flowering and that protein bodies from seeds younger than this contain only unprocessed conglutin-α.     

Oligosaccharide Side Chains of Glycoproteins that Remain in the High-Mannose Form Are Not Accessible to Glycosidases

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with α-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean α-mannosidase. (d) Treatment of the native proteins with α-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all α-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized α-mannosidase.     

In Vivo Synthesis and Turnover of alpha-Amylase in Attached and Detached Cotyledons of Vigna mungo Seeds

α-Amylase activity increased in attached cotyledons of germinated Vigna mungo seeds until the 5th day after imbibition and decreased thereafter, whereas in detached and incubated cotyledons the activity continuously increased and, at the 6th day, reached the value more than three times that of the maximum activity of attached cotyledons. Zymograms of the activities and Ouchterlony double immunodiffusion test on the activities of attached and detached cotyledons showed that the increase of activity in detached cotyledons was due to the identical enzyme as in attached tissues. α-Amylase contents, determined by single radial immunodiffusion method, changed in parallel with enzyme activity in both attached and detached cotyledons, which also suggested the de novo synthesis of α-amylase in V. mungo cotyledons.

The rate of incorporation of the label from [³H]leucine into α-amylase and the ratios of dpm in α-amylase/dpm in trichloroacetic acid-insoluble fraction did not show significant difference between attached and detached cotyledons. The results indicated that in attached cotyledons fluctuation of α-amylase activity was regulated by both synthesis and degradation of the enzyme, whereas in detached cotyledons α-amylase was synthesized and accumulated, because of low degrading activity during incubation.

     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.

     

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca²⁺ or Mg²⁺. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca²⁺ and Mg²⁺. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca²⁺ and Mg²⁺, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.     

Characterization of the mutant α-mannosidase in bovine mannosidosis

Residual acidic α-mannosidase, varying in amount up to approx. 15% of normal values, can be measured in various organs of a calf with mannosidosis. The highest specific activity and relative proportion of residual activity were found in the liver. Chromatography on DEAE-cellulose showed that the residual activity was associated with two components, which were eluted at comparable positions with those found in normal tissues. The residual activity had a lower thermal stability and a higher K_m value for a synthetic substrate than did the normal enzyme. No differences in molecular weight or electrophoretic mobility between normal acidic α-mannosidase and the residual activity were observed by gel filtration and electrophoresis on cellulose acetate respectively. The isoelectric focusing profiles for the α-mannosidase in the normal and pathological livers were very similar. It is suggested that a mutant enzyme, resulting from a mutation in a structural gene, accounts for the residual acidic α-mannosidase in mannosidosis. The mutant enzyme, which cross-reacts with antiserum raised against normal bovine acidic α-mannosidase, is present at a decreased concentration compared with the normal enzyme. There is a correlation between the concentrations of residual activity and cross-reacting material in mannosidosis. α-Mannosidase with a pH optimum of 5.75 and which is activated by Zn²⁺ was also detected in the liver of the calf with mannosidosis. However, it is probably not a product of the defective gene because addition of Zn²⁺ indicated that it was also present in normal tissues.     

Identification of the leaf vacuole as a major nitrate storage pool

Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO₃⁻ storage sink. α-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, α-mannosidase being the more suitable. Nitrate and α-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO₃⁻ than α-mannosidase during a single washing. Empirically determined corrections were used to account for NO₃⁻ movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO₃⁻ contained 58% of the total cellular NO₃⁻ and therefore represent a major NO₃⁻ storage pool.     

beta-Galactosidases in Ripening Tomatoes

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

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [³²P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase.     

Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIα (bcTopo I, bcTopo IIIα), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg²⁺ but does not possess an efficient DNA decatenation activity. bcTopo IIIα is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg²⁺ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIα are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.     

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression,Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k _cat/K _m values for oNPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.     

Endoplasmic reticulum alpha-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc₃Man₉GlcNAc₂ N-glycan is processed by α-glucosidases I and II and α1,2-mannosidase to generate Man₈GlcNAc₂. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for α-glucosidase I, α-glucosidase II catalytic subunit, and α1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41Δ, Carot2Δ, and Camns1Δ null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.     

Purification and properties of α-d-mannosidase from jack-bean meal

1. α-Mannosidase from jack-bean meal was purified 150-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn²⁺ was added during the purification to stabilize the α-mannosidase. 2. At pH values below neutrality, α-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn²⁺. Other cations, such as Co²⁺, Cd²⁺ and Cu²⁺, accelerate inactivation; an excess of Zn²⁺ again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn²⁺ nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn²⁺ reactivates the enzyme during assay. 5. It is postulated that α-mannosidase is a dissociable Zn²⁺–protein complex in which Zn²⁺ is essential for enzyme activity.     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes

Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na⁺ and K⁺ gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na⁺_o and K⁺_o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump’s voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na⁺ entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca²⁺, due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na⁺ and Ca²⁺ concentrations.     

Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study β-1,3-glucan synthase activity changes. All tissues contained an active β-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active β-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in β-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that β-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.