Subunit Mol Research Articles

Differential Regulated Interactions of Calcium/Calmodulin-Dependent Protein Kinase II with Isoforms of Voltage-Gated Calcium Channel β Subunits

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.

Innovative Kinetic and Thermodynamic Analysis of a Purified Superactive Xylanase From <I>Scopulariopsis</I> sp.

Two isoenzymes of endo-1,4-beta-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40 degrees C, pH 6.5, were 95,000 and 9900 min-1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in DeltaH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50 degrees C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (DeltaH#=306 kJ/mol) compared with HAX (DeltaH#=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (DeltaS*=-232 J/[mol.K]) was more thermostable than HAX (DeltaS*=490 J/[mol.K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.

Significance of sulfhydryl groups in the activity of urease from pigeonpea (Cajanus cajan L.) seeds.

Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480000; subunit mol. wt. 80000), with 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82+/-0.13 'accessible' sulfhydryl groups per molecule of the enzyme protein (i.e. about one 'accessible' SH group per subunit). Denatured enzyme was found to titrate for 12.1+/-0.1 SH groups per molecule (i.e. about two SH groups per subunit). Half of the 'accessible' groups react faster than the remaining at pH 8.5 as well as pH 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration of p-chloromercuribenzoate (p-CMB), N-ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were biphasic, with half of the activity lost more rapidly than the remaining half. The loss of activity with p-CMB was linearly related to the blocking of accessible SH groups. Inactivation by p-CMB is largely reversible by addition of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed.

Isolation and characterization of a newly identified type II restriction endonuclease from a local Streptomyces sp. in Taiwan.

Streptomyces chusanensis ZS-2, isolated from a soil sample in Chusan in Taiwan, was found to produce a new Type II restriction endonuclease. This restriction enzyme was designated as SchI. The purified enzyme was characterized as having a subunit mol wt of 28 kDa, and was apparently free from exonuclease activities. It cleaves the phosphodiester bond between the fourth C and the fifth G on the 5'-CCGCGG-3' sequence of DNAs, leaving a 2-nucleotide protruding end at its 3' site. This data suggests that SchI is an isoschizomer of SacII. In addition, based on the comparison between SchI and SacII regarding reaction parameters, it seems that SchI is a better choice of restriction enzyme for genetic analysis and mapping.

Cytochrome c peroxidase from Methylococcus capsulatus Bath.

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grown Desulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μmax 0.053 h−) than on (R)-propanediol (0.017 h−1) and ethanol (0.027 h−1). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.

Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.

The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication terminator protein (RTP) of subunit mol. wt 14,500. RTP has become an attractive protein with which to study the molecular mechanism of termination because its crystal structure has now been solved and the previous lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coli system. We have exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E. coli only when two RTP dimers are bound co-operatively to overlapping core and auxiliary sequences comprising the terminus. A core sequence by itself binds one dimer of RTP, but elicits no contrahelicase activity. Binding of two RTP dimers to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for termination. RTP blocks unwinding of DNA substrates containing heteroduplex regions that include the terminus and are in the size range of approximately 50 to > 1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.

Purification and characterization of mannitol dehydrogenase from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva)

As part of our studies to elucidate the mechanisms by which biotrophic plant pathogens obtain and retain carbon from their plant hosts, we have investigated the properties of the mannitol dehydrogenase (E.C. 1.1.1.67) of Cladosporium fulvum (syn. Fulvia fulva). The enzyme has been purified to near-homogeneity and has a subunit mol. wt of 29 kDa and is probably tetrameric. The Michaelis-Menten constants for mannitol and fructose are high and the equilibrium constant lies towards mannitol accumulation. A β-fructosidase (invertase) activity was detected and found to be largely wall-bound. The properties of these enzymes are consistent with a role in the hydrolysis of photosynthetically produced sucrose and the accumulation of mannitol as a storage compound during fungal growth in the plant.

Isolation of lactose-binding lectins from axolotl ( Ambystoma mexicanum)

1. 1. Lactose-inhibitable hemagglutination activity was identified in extracts of axolotl ( Ambystoma mexicanum) larvae. 2. 2. Two types of lectin were isolated from extracts by affinity chromatography on lactose-Sepharose. 3. 3. A thiol-independent lectin of subunit mol. wt 15 kDa and a thiol-dependent lectin of subunit mol. wt 18 kDa were identified. 4. 4. The 15 kDa and a 18 kDa polypeptides were weakly reactive with polyclonal anti-human galaptin serum.

Nucleotide sequence of dihydrofolate reductase type VI

The complete sequence of the type VI dihydrofolate reductase (DHFR) gene from plasmid pUK672 was determined. The structural gene coded for a polypeptide of 157 amino acids which had a deduced mol. wt of 17,424. Comparison with amino-acid sequences of the type I, type V and Escherichia coli K12 chromosomal DHFRs showed that there was 63%, 61% and 31% homology respectively. Putative RNA polymerase and ribosomal binding sites were identified proximal to the initiation codon and a feature consistent with transcription termination was present distal to the coding region. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the enzyme had a subunit mol. wt of 17,500.

Purification and characterization of rat uterine glycerylphosphorylcholine diesterase and its tissue-specific induction by 17 beta-estradiol.

The rat uterine enzyme glycerylphosphorylcholine (GPC) diesterase found in the proestrous secretions was purified and characterized biochemically with respect to its subunit mol wt, native size, pI, and amino acid and carbohydrate composition. The 30-kDa protein was assessed to have a native mol wt of 105 kDa, as determined by analytical gel filtration. It had a basic pI with an unusually high carbohydrate/protein ratio (0.83:1). The estrogen inducibility of the protein was identified by its de novo synthesis in vitro after in vivo 17 beta-estradiol administration. For this experiment, uteri from estradiol-treated immature rats were incubated in the presence of [14C]glucosamine, and the 14C-labeled total glycoconjugates were then subjected to the enzyme purification procedure. The peak enzyme fraction was observed to correspond to one of the 14C-labeled protein peaks in the elution profile of the glycoconjugates. Assay of GPC diesterase activity showed significant enhancement only in the uterus, not in other organ systems, after in vivo estradiol treatment, and this hormone-stimulated increase was inhibited by nafoxidine, a potent estrogen antagonist. The enzyme activity profile of the three main separated uterine cellular types showed that the enzyme was secreted by epithelial cells. No antigenic homology of the enzyme was observed with GPC diesterase from nonmammalian sources. The results suggested that GPC diesterase was one of the estrogen-regulated proteins of the preovulatory uterine fluid of rats, and the availability of a purified preparation of the enzyme could serve as a useful tool to study the mechanism of estrogen action.

Purification and molecular properties of glycogen phosphorylase b from mantle tissue of mussel, Mytilus galloprovincialis

1. 1. Glycogen phosphorylase b from mantle of Mytilus galloprovincialis has been purified to homogenity as judged by native and SDS-gel electrophoresis. 2. 2. The enzyme was purified 70-fold with a final spec. act. of 0.4 U/mg prot and an overall yield of 34%. 3. 3. The mol. wt of native and purified phosphorylase was estimated to be 340,000 from gel filtration studies. On SDS-gel electrophoresis, the subunit mol. wt was 86,000. The enzyme was shown to be present as a tetramer. 4. 4. Maximum activity of the enzyme was obtained at pH 7.2 and 30°C.

Significance of sulphhydryl groups in the activity of isocitrate lyase of castor endosperm

Titration of isocitrate lyase of germinating castor endosperm, a tetrameric protein (mol. wt. 140 000; subunit mol. wt. 35 000), with 5,5′-dithio-bis-(2-nitrobenzoate) (DTNB) reveals the presence of 4.15 ‘accessible’ sulphhydryl (SH) groups. Denatured enzyme is found to titrate for 7.9 SH groups per molecule. Half of the ‘accessible’ groups react more readily with DTNB than the remaining half. The enzyme is inactivated at low concentrations of p- chloromercury benzoate (p-CMB) or N- ethylmaleimide (NEM). Under the conditions [p-CMB] ⪢ [enzyme], the inactivation reaction is found to be biphasic in which half of the activity is lost more rapidly than the remaining half. With NEM (reagent in excess over enzyme), the activity is destroyed in a single exponential decay (pseudo-first order kinetics). The loss of activity is found to be linearly related to the blocking of accessible SH groups. Glyoxylate protects the enzyme strongly against attack by NEM. Protection by succinate is very much weaker. Significance of these results is discussed.

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

1. 1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively. 2. 2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn 2+, Zn 2+ and Mg 2+. The K m for Mg 2+ is lower than the physiological concentration of cation in the tissue, whereas its K m for Mn 2+ and Zn 2+ is greater than the respective in vivo concentrations. 3. 3. The joint action of Mg 2+ and Zn 2+ increases the affinity of the enzyme for the substrate Fru-1,6-P 2, though V max is reduced. 4. 4. Na + strongly inhibits the enzyme even at very low concentrations. K + has no effect whatsoever.

Antibacterial effects of different snake venoms: Purification and characterization of antibacterial proteins from Pseudechis australis (Australian king brown or mulga snake) venom

Venoms from 30 different snake species were tested in a disc diffusion assay for antibacterial effects against gram-positive and gram-negative bacteria. A number of venoms gave a zone of inhibition against both groups of bacteria, including Aeromonas hydrophila, an important pathogen of reptiles and amphibians. Two antibacterial components from the venom of an Australian elapid, Pseudechis australis (Australian king brown or mulga snake) were purified to homogeneity. The proteins, designated LAO1 and LAO2, had potent antibacterial properties associated with l-amino acid oxidase activity. Both had native and subunit mol. wts of 142,000 and 56,000, respectively. Antibacterial activity correlated with enzymatic activity and was eliminated with catalase. LAO1 and LAO2 had 244 and 113 units of l-amino acid oxidase activity/mg protein, respectively. Compared to tetracycline, a drug of choice for Aeromonas infections in humans, reptiles and amphibians, the in vitro antibacterial effects of LAO1 and LAO2 were respectively 70 and 17.5 times more effective (on a molar basis).

Purification and properties of a storage protein from the hemolymph of Rhodnius prolixus

A non-arylphorin hexameric storage protein (SPR) has been isolated from the larval hemolymph of the bug, Rhodnius prolixus. The purified protein contained 0.69% lipid and 1.04% carbohydrate. SPR had a mol. wt of 470,000, with a subunit mol. wt of 78,000. In electron micrographs, SPR molecules appeared very similar to calliphorin. After the metamorphic molt, SPR persisted at lower concentrations in the hemolymph of adult insects, and it disappeared only in fasting bugs.

Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper)

B. Lomonte, G. Rojas, J. M. Gutíerrez and G. Ramírez. Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper). Toxicon 28, 75–81, 1990.—A galactose-binding lectin, isolated from the venom of B. godmani by affinity chromatography. is an acidic protein (pI 4.9) with a subunit mol. wt of about 14,000, occurring mostly as a disulfide-linked dimer of 28,000. A small proportion of lectin appears as a monomer and as a tetramer. The lectin agglutinates erythrocytes from mice, rabbit, cow and human (all ABO types, either Rh positive or negative), but does not agglutinate horse, sheep, goat and snake ( Oxybelis aeneus, Colubridae) erythrocytes. The agglutinating activity is inhibited by 1 mM EDTA. The lectin is devoid of lethal, hemorrhagic, myotoxic, proteolytic and phospholipase A 2 activities. It is not mitogenic for human peripheral blood mononuclear cells. The only effect observed was a moderate induction of edema in the footpad of mice, with a minimal edema-forming dose of 22 μg. This effect developed rapidly, and was significantly inhibited by i.p. administration of cyproheptadine, a histamine and serotonin antagonist, before injection of the lectin. Despite the edema-forming activity observed, the low concentration of lectin in crude venom, together with its relatively low potency, suggest that this lectin is not a key component in the development of edema following envenomations by B. godmani.

Production of δ-aminolevulinate: Subcellular localization and purification of murine hepatic l-alanine: 4,5-dioxovaleric acid aminotransferase

1. 1. l-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C 57BL/6J and DBA/2J. 4. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of δ-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on l-alanine-AH-Sepharose. 8. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. 9. The yield of the purification was ca 90 μg of protein per gram liver wet weight. 10. 10. The purified enzyme had a subunit mol. wt of 146,000 ± 5000 as determined by electrophoresis under denaturing conditions.

Subunit structure and higher order assembly of the hemocyanins of five members of the naticidae family of marine gastropods: Calinaticina oldroydii (DALL), Euspira heros (SAY), Neverita Duplicata (SAY), Polinices draconis (DALL), and Polinices lewisii (GOULD)

1. 1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. 2. At pH 8.0, 0.05 M Mg 2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient ( s 20 o , w ) of 131.3 (±0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. 3. The average particle masses based on STEM measurements are 8.85 × 10 6. 13.3 × 10 6, and 17.1 × 10 6 da for the di-, tri-, and tetra-decameric assemblies hemocyanin. 4. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 × 10 5 to 4.8 × 10 5, suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. 5. The appearance of the larger hemocyanin particles in the electron micrograph support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same “collar” ends.

Liver purine nucleoside phosphorylase in Camelus dromedarius: Purification and properties

1. 1. Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyl transferase, EC 2.4.2.1) was purified to electrophoretic homogeneity from the liver of Camelus dromedarius. 2. 2. The enzyme appears to be a dimer with a 44,000 subunit mol. wt and displays non-linear kinetics with concave downward curvature in double reciprocal plots with respect to both inosine and orthophosphate as variable substrates. 3. 3. The effect of thiol compounds on the enzyme activity and of pH on kinetic parameters is reported.