Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Destruction of mixture of tri-hexa-chlorinated biphenyls by Rhodococcus genus strains

Destruction of polychlorinated biphenyls (PCBs) by strain-destructors Rhodococcus sp. B7a and Rhodococcus sp. G12a has been studied. It was shown that these strains destruct 78-95% of PCB mixture containing tri-hexa-chlorinated biphenyls. Rhodococcus destruct all components of the mixture of tri-, tetra-, penta-, and hexa-chlorinated biphenyls without accumulation of toxic chlorinated metabolites. The studied bacteria destruct PCB that are the most stable for oxidation, such as 2,5,2',5'-CB; 3,4,3',4'-CB; and 2,4,5,2',4',5'-CB. The most perspective strains are R. rubber P25, Rhodococcus sp. B7a and Rhodococcus sp. G12a whose metabolic potential can be used for biotechnological refinement of the environment from highly toxic pollutants.     

Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.     

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls

We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.     

Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes.

The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2',4',5',7'-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.     

Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.     

Studies of the influence of chloro-substituent sites and conformational energy in polychlorinated biphenyls on uroporphyrin formation in chick-embryo liver cell cultures.

Treatment of cultured chick-embryo liver cells with polychlorinated biphenyls (PCB) results in decreased uroporphyrinogen decarboxylase activity and increased uroporphyrin accumulation. In the present study we examined the effect of the chloro- or bromo-substituent sites in biphenyls (BP) on uroporphyrin accumulation in cultured hepatocytes and the three-dimensional structure of these congeners determined by molecular orbital calculations using a MNDO ('modified neglect of diatomic overlap') method. Among 20 congeners examined, those which were effective in stimulating porphyrin accumulation contained at least two Cl or Br atoms at the lateral adjacent positions in each phenyl ring, e.g. 3,4,3',4'-tetrachloro-, 2,4,3',4'-tetrachloro-, 3,4,5,3',4',5'-hexachloro- and 3,4,5,3',4',5'-hexabromobiphenyl, whereas those which contained less than two halogen atoms or more than three halogen atoms in each phenyl ring or those which contained halogen atoms at 2,2'-positions were not effective. On the basis of the conformational energy (delta E, difference from the most stable conformational energy), which is calculated as a function of the dihedral angle (theta) between the two phenyl rings, biphenyl congeners can be classified into four groups with different conformations. The conformation of active PCB was relatively flexible, whereas inactive species had a rigidly angulated conformation. Furthermore, the calculated probability of the conformation distribution for each congener indicated that the probability of co-planarity was higher for active biphenyls than for inactive congeners. These structural characteristics suggest the significance of both the chloro-substituent sites and the conformational energy reflecting the phenyl-ring twist angles in determining the inhibitory effect of PCB on uroporphyrinogen decarboxylase activity.     

Transformation of polychlorinated biphenyls by a novel BphA variant through the meta-cleavage pathway

The transformation of 20 polychlorinated biphenyls (PCBs) through the meta-cleavage pathway by recombinant Escherichia coli cells expressing the bphEFGBC locus from Burkholderia cepacia LB400 and the bphA genes from different sources was compared. The analysis of PCB congeners for which hydroxylation was observed but no formation of the corresponding yellow meta-cleavage product demonstrated that only lightly chlorinated congeners including one tetrachlorobiphenyl (2,2',4,4'-CB) were transformed into their corresponding yellow meta-cleavage products. Although many other tetrachlorobiphenyls (2, 2',5,5'-CB, 2,2',3,5'-CB, 2,4,4',5-CB, 2,3',4',5-CB, 2,3',4,4'-CB) and one pentachlorobiphenyl (2,2',4,5,5'-CB) tested were depleted from resting cell suspensions, no yellow meta-cleavage products were observed. For most of these congeners, dihydrodiol compounds accumulated as the endproducts, indicating that the bphB-encoded biphenyl-2,3-dihydrodiol-2,3-dehydrogenase is a key limiting step for further degradation of highly chlorinated congeners. These results suggest that engineering the biphenyl dioxygenase alone is insufficient for an improved removal of PCB. Rather, improved degradation of PCBs is more likely to be achieved with recombinant strains containing metabolic pathways not only specifically engineered for expanding the initial dioxygenation but also for the mineralization of PCBs.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy

Some 3'- and/or 5'-substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N-glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme. The above compounds, including the potential anti-(human immunodeficiency virus) agent, 3'-azido-2',3'-dideoxy-5-methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl-5-ethyluracil, which proved to be a poor substrate for uridine phosphorylase). The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest Ki was obtained in the case of 3'-azido-2',3'-dideoxy-5-methyluridine and the highest in the case of 2'-deoxylyxofuranosyl-5-ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The Ki for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine was 5-6 orders of magnitude higher than that for 2,2'-anhydro-5-ethyluridine. Competitive inhibition was observed in all cases. For these three molecules computer-aided molecular modelling predicts the following glycosidic torsion angles chi (O4,-C1,-N1-C2): 109 degrees for 2,2'-anhydro-5-ethyluridine, and 78 degrees and 71 degrees for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine respectively. These values are corroborated by high-resolution 13C- and 1H-NMR studies. 2'-Deoxy-5-ethyluridine is predicted to have a syn conformation with chi = 46 degrees and delta E about 2.5 kJ/mol over the minimum energy (in anti position, chi = -147 degrees). 1H and 13C data including homonuclear Overhauser enhancements complete the information about the solution conformation. Considering the Ki values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2'-anhydro-5-ethyluridine. The greater than 30 degrees deviation from the N-glycosidic torsion angle of 2,2'-anhydro-5-ethyluridine results in much higher Ki values.     

Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase.

Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM.     

Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase

1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized alpha-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, K(m) for P(i)=2.2mm, and (b) in the synthetic direction, K(m) for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (K(i)=20mum) and UDP-glucose (K(i)=60mum) were particularly potent competitive inhibitors. ATP, NADP(+) and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ;glucose excess' chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide.

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3',4'-tetrachlorobiphenyl and 2,4,5,3',4'-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3',4'-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3',4',5'-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3',4'-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.     

Inhibition of the glycogen phosphorylase system during ochratoxicosis in chickens

Graded doses of ochratoxin A incorporated into the diet (0, 0.5, 1.0, 2.0, 4.0, and 8.0 micrograms/g) of broiler chickens significantly (P < 0.05) inhibited activity of protein kinase, the initiator enzyme of the glycogen phosphorylase system, in the livers at all dose levels. Only the highest dose, 8.0 micrograms/g, significantly reduced the total activity of phosphorylase kinase, which is activated by protein kinase. The total activity of phosphorylase, which is activated by phosphorylase kinase, was unaltered by ochratoxin A at any level. Additon of ochratoxin A to liver extracts control birds inhibited protein kinase but not phosphorylase kinase. When added to extracts of livers from control birds, cyclic adenosine 3',5'-monophosphate stimulated protein kinase but not phosphorylase kinase. The cyclic adenosine 3',5'-monophosphate had no effect when added to extracts from birds fed ochratoxin A. These results suggest that ochratoxin A affects primarily the cyclic adenosine 3',5'-monophosphate-dependent protein kinase which initiates the enzymatic cascade leading to glycogenolysis. Furthermore, these results conform an earlier assignment on morphological criteria of the glycogenosis of ochratoxicosis as a type X glycogen storage disease.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Regulation of the dephosphorylation of phosphorylase A by glucose, AMP and polyamines

1. The effect of glucose, caffeine, AMP and polyamines was investigated on the dephosphorylation of phosphorylase a by the catalytic subunits of protein phosphatase-1 and -2A. 2. Caffeine at 1-20 microM inhibited the dephosphorylation of the dimeric phosphorylase a at 37 degrees C using skeletal muscle enzymes; 0.1-10 mM of caffeine enhanced the rate of dephosphorylation greatly at 13 degrees C and slightly at 37 degrees C. 3. alpha-D-Glucose was more effective in accelerating both the dephosphorylation and the tryptic digestion of phosphorylase a than the beta-anomer. 4. Polyamines were found to moderate the inhibitory effect of AMP at concentrations which may occur in the tissues. In the presence of 5 mM glucose polyamines could cancel the AMP inhibition of the dephosphorylation of liver phosphorylase a by hepatic protein phosphatase-1 and -2A.