Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated (Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.     

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.     

Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10(-6)). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Azithromycin and clarithromycin inhibition of 50S ribosomal subunit formation in Staphylococcus aureus cells

A mercapturic acid attached to the aromatic ring of toluene was for the first time detected in human urine as a metabolite of toluene. Since the metabolism of toluene is usually considered to take place at the side-chain, this gives, besides the biosynthesis of cresols, a further hint of a metabolic conversion of the aromatic system. We examined a group of 33 workers occupationally exposed to toluene, determining the concentrations of toluene in ambient air and in whole blood, o-cresol and hippuric acid in urine and p-toluylmercapturic acid (p-TMA) in urine. All blood and urine samples were collected post-shift. The renal excretion of S-p-toluylmercapturic acid showed highly significant correlations with established parameters of a biological monitoring of toluene. The median ambient air concentration was 63 ppm, ranging from 13 to 151 ppm, the median concentration of toluene in whole blood was 804 microg/l, corresponding to median urinary concentrations for o-cresol of 2.3 mg/l, hippuric acid of 2.3 g/l and p-TMA of 20.4 microg/l. p-TMA was not detectable in urine samples of a control group of 10 non-exposed persons. Both the German Biological Tolerance Values (BAT-values) for toluene in blood (1000 microg/l) and o-cresol in urine (3 mg/l) correspond to a mean p-TMA elimination of approximately 50 microg/l, and thus are in agreement with each other. According to our results p-TMA reflects internal toluene exposure diagnostically sensitive and specifical. With the developed analytical procedure we determined a median benzylmercapturic acid (BMA) concentration of 190 microg/l in the urine samples of the toluene exposed persons. We also determined a median BMA concentration of 30 microg/l in the control samples of non-exposed persons. However, these results are preliminary and require further confirmation as the reliability of the method was determined only for p-TMA.     

Purification and subunit structure of extracellular superoxide dismutase from mouse lung tissue

The first purification of mouse extracellular superoxide dismutase (EC-SOD) and the analysis of the native enzyme are described. Mouse EC-SOD was purified from lung tissues with a high recovery (41%) and a specific polyclonal antibody against the purified enzyme was obtained. The purified enzyme had a strong affinity for, heparin and a molecular mass of 150 kDa (estimated by a gel filtration chromatography). The native mouse EC-SOD was composed of two different sizes of subunits, a M(r) of 33 and 35 kDa (determined by SDS-PAGE). The 35-kDa subunit had an interchain disulfide bond at the C-terminus and existed as a covalent dimer in the molecule, whereas the 33-kDa subunit resulted from the 35-kDa subunit by truncating its C-terminus as a posttranslational modification, with resultant loss of the interchain disulfide bond. These results suggest that the native mouse EC-SOD is a heterotetramer composed of two different dimers, with or without a covalent bond.     

Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I

Type I restriction-modification (R-M) enzymes are composed of three different subunits, of which HsdS determines DNA specificity, HsdM is responsible for DNA methylation and HsdR is required for restriction. The HsdM and HsdS subunits can also form an independent DNA methyltransferase with a subunit stoichiometry of M2S1. We found that the purified Eco R124I R-M enzyme was a mixture of two species as detected by the presence of two differently migrating specific DNA-protein complexes in a gel retardation assay. An analysis of protein subunits isolated from the complexes indicated that the larger species had a stoichiometry of R2M2S1and the smaller species had a stoichiometry of R1M2S1. In vitro analysis of subunit assembly revealed that while binding of the first HsdR subunit to the M2S1complex was very tight, the second HsdR subunit was bound weakly and it dissociated from the R1M2S1complex with an apparent K d of approximately 2.4 x 10(-7) M. Functional assays have shown that only the R2M2S1complex is capable of DNA cleavage, however, the R1M2S1complex retains ATPase activity. The relevance of this situation is discussed in terms of the regulation of restriction activity in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.     

Isolation and characterization of a two-subunit cytochrome b-c1 subcomplex from Rhodobacter capsulatus and reconstitution of its ubihydroquinone oxidation (Qo) site with purified Fe-S protein subunit

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.     

Purification and G protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.     

Targeting of the catalytic subunit of protein phosphatase-1 to the glycolytic enzyme phosphofructokinase

In this study, we demonstrate that the catalytic subunit of rabbit muscle protein phosphatase-1 (PP1) binds to muscle phosphofructokinase (6-phosphofructo-1-kinase, PFK). A protein of 85 kDa was isolated from rat muscle by affinity chromatography on PP1-Sepharose and was identified as phosphofructokinase by partial amino acid sequence analysis. This novel finding of a protein-protein interaction between PP1 and PFK was confirmed by reciprocal experiments in which the binding of PP1 to PFK-agarose was demonstrated. Elution of PP1 from PFK-agarose was maximal at ca. 0.4 M NaCl. The specificity of binding was demonstrated by isolation of PP1 from a partially purified rabbit muscle PP1 preparation. All four known isoforms of PP1 (PP1alpha, PP1gamma1, PP1gamma2, and PP1delta) were shown to bind to PFK-agarose. The activity of PP1 was only partially inhibited by PFK. The preformed complex between PP1 and PFK did not bind to inhibitor-2-Sepharose. The stoichiometry of binding of PP1 to the PFK monomer was found to be 1:1 in the isolated PP1.PFK complex. An interaction between PP1 and PFK in muscle extracts was demonstrated by their coimmunoprecipitation. Our findings raise the interesting possibility that PP1 may be targeted to PFK, and may be physiologically relevant in the context that PFK and other glycolytic enzymes have been shown to be micro-compartmentalized by binding to F-actin. This in turn points to a role for PP1 in control of glycolytic flux by protein phosphorylation-dephosphorylation mechanisms.     

Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes

Previous studies have shown that toluene, which is commonly abused, depresses neuronal activity and causes behavioral effects in both animals and man similar to those observed for ethanol. In this study, the oocyte expression system was used to test the hypothesis that toluene, like ethanol, inhibits the function of ionotropic glutamate receptors. Oocytes were injected with mRNA for specific N-methyl-D-aspartate (NMDA) or non-NMDA subunits and currents were recorded using conventional two-electrode voltage clamp. To enhance the low water solubility of toluene, drug solutions were prepared by mixing toluene with alkamuls (ethoxylated castor oil) at a 1:1 ratio (v:v) and diluting this mixture to the appropriate concentration with barium-containing normal frog Ringer solution. Alkamuls, up to 0.1%, had no significant effects on membrane leak currents or on NMDA-induced currents. Toluene, up to approximately 9 mM, had only minor effects on membrane leak currents but dose-dependently inhibited NMDA-mediated currents in oocytes. The inhibition of NMDA receptor currents by toluene was rapid, reversible and the potency for toluene's effects was subunit dependent. The NR1/2B subunit combination was the most sensitive with an IC50 value for toluene-induced inhibition of 0.17 mM. The NR1/2A and NR1/2C receptors were 6- and 12-fold less sensitive with IC50 values of 1.4 and 2.1 mM, respectively. In contrast, toluene up to approximately 9 mM did not inhibit kainate-induced currents in oocytes expressing GluR1, GluR1(+)R2 or GluR6 subunits. These results suggest that some of the effects of toluene on neuronal activity and behavior may be mediated by inhibition of NMDA receptors.     

Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase

In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N-terminal sequence of residues 1-38 is involved in activation of PP1c (kcat) and the ankyrin repeats (residues 39-295) were involved in substrate binding (Km). The ankyrin repeats alone (residues 39-295) and the C-terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17-295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N-terminal sequence (residues 1-38) and the ankyrin repeats are involved in binding PP1c. The C-terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N-terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.     

Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase

Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.     

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Improved purification, physical properties and subunit arrangement in purified polymers

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.     

Sequence of the fifth largest subunit of RNA polymerase II from plants

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.     

Effect of deletion from the carboxyl terminus of the 12 S subunit on activity of transcarboxylase

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. The central hexameric 12 S subunit of the enzyme associates with six 6 S subunits in the complete enzyme complex. We have constructed a series of cloned genes which encode COOH-terminal truncations of the 12 S subunit. Five of these subunits, which remained soluble following expression in Escherichia coli and were missing from 39 to 97 COOH-terminal amino acids, were purified and compared to the full-length subunit after enzyme complexes were assembled in vitro. All of the truncated subunits were 90% as active in the transcarboxylase reaction as wild type except the reaction containing the shortest complex, TC-12 S (1-507), which had 54% of the wild type activity (TC-12 S-WT). The reduced activity was not due to a lack of CoA ester binding sites or the Km for substrate. However, TC-12 S (1-507) was slower to form than TC-12 S-WT and had more incomplete complexes as judged by high performance liquid chromatography gel filtration profiles and electron microscopy. Isolated TC-12 S (1-507) was 70-80% as active as TC-12 S-WT. We also noted that the truncated form was heat-labile compared to wild type. We conclude that the COOH-terminal region of the 12 S subunit plays a role in assembly and stability of the hexamer and also affects the binding of 6 S subunits to form enzyme complexes. Once complexes do form, the catalytic capacity of TC-12 S (1-507) is almost the same as TC-12 S-WT.     

Enhancement of Aromatic Hydrocarbon Biodegradation by Toluene and Naphthalene Degrading Bacteria Obtained from Lake Sediment: The Effects of Cosubstrates and Cocultures

Toluene and naphthalene degrading (ND) bacteria, obtained from contaminated lake sediment, were used to degrade both monoaromatics and polycyclic aromatic hydrocarbons (PAHs) and the effects of cosubstrates and cocultures were examined. When toluene and naphthalene enrichments were used, the effect of the substrate interaction on their metabolism was found to be inhibitory and yet the cocultures were stimulatory, especially for toluene enrichment degradation of naphthalene (with toluene). Pseudomonas putida M2T14, a toluene degrading isolate, could efficiently degrade benzene and toluene but not naphthalene. Nonetheless, when toluene was present, this monoaromatic degrader became capable of degrading PAHs, among which the methyl substituted PAHs (mPAHs) were preferred to their corresponding unsubstituted PAHs (uPAHs). Pseudomonas azelaica ND isolate could degrade benzene, toluene, and all test PAHs. Although the uPAHs were preferred, the degradation rates of mPAHs were greatly increased via substrate interactions with naphthalene. The interaction modes of dual aromatic hydrocarbons (AHs) degraded by P. putida M2T14 and P. azelaica ND were cometabolism, synergism, no effect, inhibition, and antagonism. However, when a negative effect of biodegradation from the interaction of these AHs was found on one isolate, a positive effect would be found on the other. When benzene was present, it exhibited inhibitory effects on aromatic hydrocarbon biodegradation by M2T14 and ND isolates. A study of the biodegradation of the ternary mixture of benzene, toluene, and naphthalene by both isolates together illustrated that not only was inhibition relieved but that degradation of each compound was also greatly enhanced. Degradation by the toluene and the ND bacteria could be facilitated by complementary substrate interactions between monoaromatics and PAHs and by bacterial association. These model organisms may be very useful for the study of complex aromatic hydrocarbon degradation and for bioremediation purposes.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.     

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.