Subunit Mr Research Articles

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase)

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of β-mercaptoethanol (β-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of β-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.

Simultaneous separation of taxon-specific crystallins from Mule duck and characterization of their enzymatic activities and structures.

Methods to obtain pure proteins in large amounts are indispensible in protein research. We report here a large-scale/simultaneous isolation of taxon-specific crystallins (ɛ- and δ-crystallin) from the eye lenses of Mule duck. We also investigate the compositions, enzymatic activities, and structures of these purified taxon-specific proteins. A relatively mild method of ion-exchange chromatography was developed to fractionate ɛ-crystallin and δ-crystallin in large amount, ca. ∼6.60mg/g-lens and ∼41.0mg/g-lens, respectively. Both crystallins were identified by electrophoresis, HPLC, and MALDI-TOF-MS. ɛ-Crystallin, with native composition of Mr 142kDa, consisted of two subunits of 35kDa and 36kDa, while δ-Crystallin, with native molecular mass of 200kDa, comprised single subunit of Mr ∼50kDa. Both ɛ- and δ-crystallin were tetramers. The former showed lactate dehydrogenase (LDH) activity, while the latter appeared slightly active in an argininosuccinate lyase (ASL) assay. Raman spectroscopic results indicated that the secondary structures of ɛ- and δ-crystallin were predominantly α-helix as evidenced by the vibrational stretching of amide III over 1260cm-1 and amide I at 1255cm-1, in greatly contrast to the anti-parallel β-sheet of α- and β-crystallin as demonstrated by amide III at 1238cm-1 and amide I at 1672cm-1. The microenvironments of aromatic amino acids and the status of thiol groups also vary in different crystallins. The compositions, enzyme activities, and structures of the ɛ- and δ-crystalline of Mule duck are different from those of Muscovy duck (Cairina moschata) or Kaiya duck (Anas Platyrhynchos var. domestica), which reflect faithfully species specificity.

The Cstf2t Polyadenylation Gene Plays a Sex-Specific Role in Learning Behaviors in Mice.

Polyadenylation is an essential mechanism for the processing of mRNA 3′ ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNA-binding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction.

A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties

An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60-80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-D-galactopyranoside, α-D-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-D-galactoside and 12 mM versus α-D-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg(2+) and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of -SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.

High Molecular Weight Glutenin Subunits in Some Durum Wheat Cultivars Investigated by Means of Mass Spectrometric Techniques

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars (Simeto, Svevo, Duilio, Bronte, and Sant'Agata), largely cultivated in the south of Italy, and of 13 populations of the old spring Sicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS). M(r) of the intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same two HMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS 1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolated HMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively. Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, with differences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acid substitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between the M(r) of the 1Bx subunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and is consistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.

Adenosine deaminase from Streptomyces coelicolor: Recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr = 48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s −1at 30 °C. Since adenine is deaminated ∼10 3 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β- d-arabinofuranosyl-adenine (ara-A), 5′-AMP, 5′-ADP and 5′-ATP, are not substrates for the enzyme. Coformycin and 2′-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.

Expression, Purification, and Characterisation of Dehydroquinate Synthase fromPyrococcus furiosus

Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and kcat 3.0 sec−1 at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica

Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38 °C and pH 7.8, specific activity values were 10.2 U mg − 1 and 64.5 U mg − 1 , with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46 ± 8 μM (DTNB) and 30 ± 5 μM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated.

FRET-Based Mapping of Three Structural Domains within the Full-Length Ryanodine Receptor Type 1

The type 1 ryanodine receptor (RyR1) mediates excitation contraction coupling in skeletal muscle by releasing intracellular calcium in response to cell depolarization. Understanding the structure and conformational dynamics of RyR1 is challenging due to its size (homotetrameric subunit Mr∼560 kDa) and its association with numerous regulatory proteins. However, recent X-ray crystallographic studies indicate that a fragment comprised of the first 200 amino acids of RyR1 folds into a compact β-trefoil domain. Based on structural homology with the inositol trisphosphate receptor, the next 400 amino acids of RyR1 are predicted to fold into a second β-trefoil and an alpha helical domain. In this report, the relative orientation of these three domains (termed here as β1, β2 and α1) within the full-length RyR1 protein has been investigated using a novel FRET-based technique. This method monitors the relative proximity of a GFP fluorescent donor fused into RyR1 and a fluorescent acceptor, Cy3NTA, targeted to poly-histidine (His) tags inserted into the primary sequence of RyR1. In this study, GFP was fused to single positions within β1, β2 and α1 and FRET was then measured to Cy3NTA targeted to 6 His tags singly introduced into these three domains. The results of these measurements provide a detailed picture of the relative orientation of these three sub-domains within full-length RyR1 expressed in a cellular context. Further structural studies using this technique can now be undertaken to determine how these domains move during channel gating. (Supported by NIH grant R21ARO56406).

Native polymeric forms of properdin selectively bind to targets and promote activation of the alternative pathway of complement

Properdin, a positive regulator of the complement system, has recently been reported to bind to certain pathogenic microorganisms, to early or late apoptotic and necrotic cells, and to particular live human cell lines, thus providing a platform for de novo convertase assembly and complement activation. These studies, with some contradictory results, have been carried out with purified properdin, which forms a series of oligomers of a ∼53,000 Mr subunit, assembling into dimers (P 2), trimers (P 3), tetramers (P 4) and higher forms (P n). The P n forms have been shown to likely be an artefact of purification that results from procedures including freeze-thawing of properdin. In this study we isolated the individual natural forms of properdin (P 2, P 3, and P 4) and separated them from the P n forms present in purified frozen properdin using ion exchange and/or size exclusion chromatography. We analyzed the ability of each form to bind to live or necrotic Jurkat and Raji cells, rabbit erythrocytes (E R), and zymosan by FACS analysis. While the unseparated properdin and the purified P n forms bound to all the surfaces except E R, the physiological P 2–P 4 forms specifically bound only to zymosan and to necrotic nucleated cells. Our results indicate that aggregated P n present in unseparated properdin may bind non-specifically to some surfaces and should be separated before analysis in order to obtain meaningful results. Finally, we have determined for the first time that the physiological forms of human properdin can selectively recognize surfaces and enhance or promote complement activation, which is in agreement with the reported role for properdin as a complement initiator.

Isolation, purification and characterization of an N-acetyl-D-lactosamine binding mitogenic and anti-proliferative lectin from tubers of a cobra lily Arisaema utile Schott

Lectins are the carbohydrate-binding proteins of non-immune origin which have been the subject of intense investigation over the last few decades owing to the variety of interesting biological properties. Most of the lectins which have been purified and characterized from plants have been obtained from dicotyledons. In the present study a lectin was purified from tubers of a monocot plant Arisaema utile (AUL) Schott by affinity chromatography on asialofetuin-linked amino activated silica beads. AUL gave a single band in SDS-PAGE at pH 8.3 corresponding to subunit Mr 13.5 kDa. The native molecular mass of AUL was 54 kDa suggesting a homotetrameric structure. AUL gave multiple bands in isoelectric focusing and in native PAGE at pH 8.3. AUL was inhibited by N-acetyl-D-lactosamine (Lac NAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. When treated with denaturing agents, the lectin was stable in the presence of urea (3 M), thiourea (4 M) and guanidine HCl (4 M). AUL was a glycoprotein with a carbohydrate content of 1.2%. Complete loss of activity was observed upon modification of tryptophan residues of the lectin. The activity was reduced to 25% after modification of tyrosine. Chemical modification of arginine, histidine, serine and cysteine residues of AUL did not affect its activity. Using Far UV CD spectra the estimated secondary structure was 37% α-helix, 25% β-sheet and 38% random contributions. The lectin showed potent mitogenic response towards human lymphocytes. In vitro anti-proliferative assay using 11 human cancer cell lines resulted in 50% inhibition of six cell lines viz. SW-620, HCT-15, SK-N-SH, IMR-32, Colo-205 and HT-29 at 38, 42, 43, 49, 50 and 89 µg/ml, respectively.

What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

Larvicidal Effects of a Chitin-Binding Vicilin from Erythrina velutina Seeds on the Mediterranean Fruit Fly Ceratitis capitata

Chitin-binding vicilin from Erythrina velutina seeds was purified by ammonium sulfate followed by affinity chromatography on a chitin column and gel filtration on Superose-6-10-300-GL. The Erythrina velutina vicilin, called EvV, is a tetrameric glycoprotein composed of 1.85% carbohydrates and M r of 216.6 kDa, consisting of two subunits of M r of 54.8 and two subunits of M r of 50.8 kDa. The EvV homogeneity was confirmed in native PAGE where it was observed to be a unique acid-protein band with slow mobility in this gel. Effect of EvV on C. capitata larvae was examined by bioassay and its mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV when added to diet caused strong effect on mortality (ED50 of 0.14%) and larval mass (WD50 of 0.12%). These deleterious effects were associated to the binding to chitin structures present in peritrophic membrane and to gut epithelial cells, and its low digestibility in C. capitata digestive tract. These results are the first demonstration of a proteinaceous bioinsecticide from plant origin effective against C. capitata larvae. EvV may be part of the pest management programs or an alternative in plant improvement program.

Purification and characterization of type II collagen from chick sternal cartilage

Type II collagen was purified from sternal cartilage of the chick using a combination of pepsin digestion, NaCl precipitation and DEAE-sepharose CL 6B ion exchange chromatography. Pepsin-solubilized type II collagen of higher stability can be obtained with the extraction time of 32h, 0.5% pepsin concentration at 20°C. The purified preparation showed a single peak on RP-HPLC and a single band (α-chain) and its dimers (β-chains) on SDS–PAGE with a subunit Mr of 110kDa. The amino acid composition of the type II collagen derived from chick cartilage was closer to that of reference Sigma–Aldrich type II collagen which contains more imino acid. Analysis by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) revealed that type II collagen from chick sternal cartilage retains more intermolecular crosslinks during the purification process. Collagen purified from chick sternal cartilage was typical type II collagen and may find applications in functional foods.

Effects of a Chitin-Binding Vicilin fromEnterolobium contortisiliquumSeeds on Bean Bruchid Pests (Callosobruchus maculatusandZabrotes subfasciatus) and Phytopathogenic Fungi (Fusarium solaniandColletrichum lindemuntianum)

Chitin-binding vicilin from Enterolobium contortisiliquum seeds was purified by ammonium sulfate followed by gel filtration on Sephacryl 300-SH and on Sephacryl 200-SH. The vicilin, called EcV, is a dimeric glycoprotein composed of 1.03% carbohydrates and a Mr of 151 kDa, consisting of two subunits of Mr of 66.2 and 63.8 kDa. The EcV homogeneity was confirmed in a PAGE where it was observed to be a unique acid protein band with slow mobility in this native gel. E. contortisiliquum vicilin (EcV) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae and for phytopathogenic fungi, F. solani and C. lindemuntianum. EcV was very effective against both bruchids, producing 50% mortality for Z. subfasciatus at an LD50 of 0.43% and affected 50% of the larvae mass with an ED50 of 0.65%. In artificial diets given to C. maculatus, 50% of the larvae mass was affected with an ED50 of 1.03%, and larva mortality was 50% at LD50 of 1.11%. EcV was not digested by midgut homogenates of C. maculatus and Z. Subfasciatus until 12 h of incubation, and at 24 h EcV was more resistant to Z. subfasciatus larval proteases. The binding to chitin present in larvae gut associated to low EcV digestibility could explain its lethal effects. EcV also exerted an inhibitory effect on the germination of F. solani at concentrations of 10 and 20 microg mL-1. The effect of EcV on fungi is possibly due to binding to chitin-containing structures of the fungal cell wall.

The interaction of the 11S globulin-like protein of kiwifruit seeds with pepsin

In a search for aspartic proteinase inhibitors (APIs) in kiwifruit seeds, we observed pepsin inhibitory activity (PIA) in an abundant globulin fraction extracted in high salt buffer with a Mr of ∼148 kDa by gel-filtration. On a SDS-polyacrylamide gel, a major protein band of 54 kDa was observed under non-reducing conditions. This band was largely replaced by two subunits of Mr 33.5 and 20 kDa under reducing conditions. N-terminal sequencing of the smaller subunit, which had associated PIA, revealed a β-subunit of the 11S globulin-like protein (11S-GLP), legumin. After trypsin, chymotrypsin or papain digestion, the α-subunit of the kiwifruit legumin (11S-GLP) was degraded to varying degrees but there was no effect on the β-subunit, or on the PIA. This 11S-GLP also appeared to inhibit bovine spleen cathepsin D, Candida albicans secreted aspartic proteinases (SAPs) 1, 2 and 4, the plant fungus Glomerella cingulata SAP as well as apple seed aspartic proteinase, but to a much lesser extent kiwifruit seed aspartic proteinase. Through kinetic analysis of pepsin inhibition, the 11S-GLP and the purified β-subunit were found to fit a Michaelis–Menten model for competitive inhibition rather than a tight-binding model characteristic for typical proteinase inhibitors. Extrapolated complete inhibition was only obtained at an 11S-GLP concentration ∼900 times that of the pepsin. Further investigation revealed that the 11S-GLP and the β-subunit acted as weak alternative substrates for pepsin. As the 11S-GLP or the β-subunit were degraded, the PIA activity declined in parallel. We discuss these results in terms of the substrate recognition site of pepsin compared with other proteinases.

Physiological Consequences of Loss of Allosteric Activation of Yeast NAD+-specific Isocitrate Dehydrogenase

Based on allosteric regulatory properties, NAD+-specific isocitrate dehydrogenase (IDH) is believed to control flux through the tricarboxylic acid cycle in vivo. To distinguish growth phenotypes associated with regulatory dysfunction of this enzyme in Saccharomyces cerevisiae, we analyzed strains expressing well defined mutant forms of IDH or a non-allosteric bacterial NAD+-specific isocitrate dehydrogenase (IDHa). As previously reported, expression of mutant forms of IDH with severe catalytic defects but intact regulatory properties produced an inability to grow with acetate as the carbon source and a dramatic increase in the frequency of generation of petite colonies, phenotypes also exhibited by a strain (idh1Deltaidh2Delta) lacking IDH. Reduced growth rates on acetate medium were also observed with expression of enzymes with severe regulatory defects or of the bacterial IDHa enzyme, suggesting that allosteric regulation is also important for optimal growth on this carbon source. However, expression of IDHa produced no effect on petite frequency, suggesting that the intermediate petite frequencies observed for strains expressing regulatory mutant forms of IDH are likely to correlate with the slight reductions in catalytic efficiency observed for these enzymes. Finally, rates of increase in oxygen consumption were measured during culture shifts from medium with glucose to medium with ethanol as the carbon source. Strains expressing wild-type or catalytically deficient mutant forms of IDH exhibited rapid respiratory transitions, whereas strains expressing regulatory mutant forms of IDH or the bacterial IDHa enzyme exhibited much slower respiratory transitions. This suggests an important physiological role for allosteric activation of IDH during changes in environmental conditions.

Purification and characterization of glutamate decarboxylase from rice germ

Glutamate decarboxylase (EC 4.1.1.15, GAD) is a pyridoxal 5′-phosphate (PLP) dependent enzyme, which catalyses the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid. GAD was purified 186-fold from rice germ using a combination of ammonium sulfate fractionation, DEAE-Sepharose FF ion exchange chromatography, Superdex-200 gel filtration chromatography, and Glu-Sepharose CL 4B affinity chromatography. The purified preparation showed a single peak on SE-HPLC with an approximate molecular mass of 78 kDa and a single band on SDS–PAGE with a subunit Mr of 40 kDa. This indicated that the GAD from rice germ existed as a dimer of homological subunits. Rice germ GAD has an optimum pH range between 5.5 and 5.8, and an optimum temperature at 40 °C. K m values for glutamic acid and PLP were determined at 32.3 mM and 1.7 μM, respectively. Chemicals reagents such as HgCl 2, KI and AgNO 3 decreased the enzyme activity by 68.5%, 44.9% and 32.4%, respectively, but 500 μM of CaCl 2 at the optimum pH could increase the activity by 145%.

Nuclear Localization of STAT5A Modified with O-Linked N-Acetylglucosamine and Early Involution in the Mammary Gland of Hirosaki Hairless Rat

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

Human Mitochondrial ClpP Is a Stable Heptamer That Assembles into a Tetradecamer in the Presence of ClpX

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.