Lynch, Thomas J.

Ross, Mark S.

English

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 39 Article 421985Demonstration of a Heat-Stable Cyclic GMPPhosphodiesterase in the Medium of PhysarumflavicomumThomas J. LynchUniversity of Arkansas at Little RockMark S. RossUniversity of Arkansas at Little RockFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Biochemistry CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the ArkansasAcademy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationLynch, Thomas J. and Ross, Mark S. (1985) "Demonstration of a Heat-Stable Cyclic GMP Phosphodiesterase in the Medium ofPhysarum flavicomum," Journal of the Arkansas Academy of Science: Vol. 39 , Article 42.Available at: http://scholarworks.uark.edu/jaas/vol39/iss1/42Arkansas Academy of Science Proceedings, Vol. XXXIX,1985 139Arkansas Academy of ScienceTable 2. Skeletal characteristics and standardized discriminant func-tion coefficients that maximize the discrimination among 68 male andfemale Black-capped and Carolina Chickadees from the Central Plainsand Ozark Plateaus regions.Discriminant functionFirst SeconSkeletal characteristic d:h 0.75 -0.25igth 0.42 -0.36I -1.02 0.81irpus length 0.25 0.46i 0.15 0.68ridth 0.47 -0.40Femur lengthHumerus lenUlna lengthCarpometacarpusFIRST DISCRIMINANTFUNCTION Keel lengthFigure. Ordination with respect to the first two discriminant functionsof 68 skeletal specimens ofBlack-capped and Carolina Chickadees.(Darkened symbols represent Black-capped Chickadees, open onesCarolina Chickadees. Squares are male birds, circles, females. Arrowsindicate the specimens in question.)Synsacrum widtin the smallest group. Because we had only 14 female Black-capped and Carolina Chickadees, it was necessary to a priorireduce the number ofvariables used to 13. Only the 15 variables that showed univariate significance were considered (Table 1), and from these culmen depth and scapulalength were omitted (the former because it is difficult to measure accurately, the latter because of great variation within groups). The 13 variableswere used in a step-wise DFA, using the criterion ofmaximizing the Mahalonobis distance among the 4 groups (males and females of both Black-capped and Carolina Chickadees). The infrequent missing values were estimated using the mean values for the appropriate group (e.g., Carolinafemale). The whole analysis was repeated using 75 chickadees by adding specimens from a wider geographical range. The results of the two analyseswere essentially similar to those described below, which are based on the one using the 68 specimens.The step-wise DFA identified 6 variables that maximized the separation among the 4groups of chickadees. These variables, and their standar-dized discriminant functions coefficients are listed in Table 2. As can be seen from the Figure, the two species are separated along the first discrimi-nant function axis (DF-1), accounting for 82.4% of the variance among the four groups.Ulna length is the variable that contributes most to the separation between species with femur length and synsacrum width being of secondaryand tertiary importance (Table 2). In total, Black-capped Chickadees, which have relatively large positive values on DF-1, have a combinationof a relatively short ulna, long femur and humerus, and wide synsacrum. The measures of wing length, other than ulna length, interestingly arepositively correlated to DF-1. The second DF (accounting for an additional 17.4% of the variation among the groups) separates male and femaleBlack-capped Chickadees but not the sexes of Carolina Chickadees (Figure). Ulna length, again, is the most important variable on the axis, withmale atricapillus having longer ulnae and keels, but relatively narrower synsacra and shorter humerae than females (Table 2).Inevaluating the precision of the discriminat function analysis, 82% of the specimens (56 out of 68) were correctly categorized as to groupaffinity. Eleven of the misidentifications were withregard to sex within species, and only one specimen was grouped with the wrong species. Thiswas the female Black-capped Chickadee from Phillips Co., Kansas (KU 61722 9 )> tnat is clustered with the Carolina Chickadees in the Figure.The analysis identified it as a female Carolina Chickadee. Thus 99% of the specimens were correctly positioned with respect to species.The two specimens in the Kansas collection that were suspect as toidentification were both identified by the analysis as being Carolina Chickadees(marked by the two arrows in the Figure). As is shown in the Figure, the male specimen clearly is a Carolina Chickadee whereas the female specimenis close to the misclassified Black-capped Chickadee. Infact this female bird was identified with a rather high probability ofbeing a Carolina Chickadee.Ithad a 69% probability ofbeing a female Carolina (which it is), and 26% probability of being a male Carolina, which leaves only a 5% probabilityof being a Black-capped Chickadee.Insummary, Black-capped and Carolina Chickadees are readily identified from skeletal material and two suspect skeletal specimens labeledBlack-capped Chickadees from Arkansas were indeed Carolina Chickadees. Thus, there are still no records of the Black-capped Chickadee in Arkansas.(In completing this study, Rising's contribution was supported by the Natural Sciences and Engineering Research Council of Canada. JimDick of the Royal Ontario Museum, and Marion Mengel and Steven Johnson, of the University of Kansas Museum of Natural History, were veryhelpful inmaking specimens available. Thanks are due Janet Mannone for managing the data. J. D. Black was especially helpful for his comments.)DOUGLASJAMES, Department of Zoology, University ofArkansas, Fayetteville, AR 72701, and JAMES D.RISING, Department of Zoology,University of Toronto, Toronto, Ontario M53 IA1, Canada.DEMONSTRATION OF A HEAT-STABLE CYCLIC GMP PHOSPHODIESTERASEINTHE MEDIUMOF PHYSARUM FLA VICOMUMAlthough the role ofcyclic AMPin cellular regulation has been well characterized during the last two decades, the function ofcyclic guanosinemonophosphate (cyclic GMP) remains vague. Cyclic GMP phosphodiesterase is the enzyme responsible fordegrading cyclic GMP to 5 'GMP andthus is of major importance in maintaining cellular levels of cyclic GMP. We have identified an extracellular cyclic GMP phosphodiesterase in139Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42Published by Arkansas Academy of Science, 1985Arkansas Academy of Science Proceedings, Vol. XXXIX,1985140General NotesTable 1. Effect of Heat on Cyclic GMP Phosphodiesterase Activity.nMole/min/ml % ActivityUnheated Heated RemainingStarting Medium 146 86 60%30-80% Pellet 160 134 84%Samples of starting medium (sm) and ammonium sulfate 30-80% pelletwere assayed for cGMP phosphodiesterase activity without heating(unheated) and after a 5 minexposure to a boiling water bath (heated).The sm is an average of four experiments and the 30-80% pellet is anaverage of three experiments.Table 2. Effect of Various Compounds on Cyclic GMPPhosphodiesterase Activity.Additive Concentration ijMole/min/ml % ActivityControl 0 162 100%MIX 7 MM 50 31%Theophylline 10 MM 63 39%Caffeine 10 MM 94 58%Chlorpromazine 500 pM 207 128%Chlorpromazine 1 MM 207 128%Compound 48/80 10 pG 253 156%Compound 48/80 100 jjg 225 139%Dialysed samples ofthe ammonium sulfate 30-80% pellet were assayedin the presence of the above indicated reagents. MIXis 3-isobutyl-l-methylxanthine.Table 3. Effect of pH on cGMP Phosphodiesterase Activity.Starting Medium 30-80% pelletpH nMole/m in/ml % Activity nFjole/m in/ml % Activity11% 56 712<4157bl XZ5 7%Z 659 79%6 125100% 830 100%7 22!;<m 1678 20%93Cyclic GMP phosphodiesterase was determined at the pH values in-dicated. A citrate buffer was used for pH 4 and 5, Pipes buffer forpH 6 and 7, and Tris for pH 8.0.Figure 1. Samples of starting medium (SM) and 30-80% ammoniumsulfate pellets were assayed for cyclic GMP phosphodiesterase activityafter being heated in a boiling water bath for the times indicated onthe abscissa.ASSAY TIME (min)Figure 2. Cyclic GMP phosphodiesterase activity inheated and unheatedsamples was measured by paper chromatography. The 30-80% am-monium sulfate pellet for heat treatment was placed in a boiling waterbath for 10 min and then returned to 30 °C. Reaction mixtures of 300fi\ each were prepared for heat-treated and non-heat treated samples.Duplicate 0.025 ml samples were withdrawn at the indicated times andadded to tubes containing 0.005 ml of 2 MHC1 to stop the reaction.Cold 5'GMP (0.005 ml of 20 mM solution) was added and aliquotswere spotted on Whatman filter paper. Descending paperchromatography was done overnight and the 3H-5'GMP spots cut outand counted.the medium from the plasmodium of the myxomycete Physarum flavicomum. This cyclic GMP phosphodiesterase appears to be very similar tothe previously reported extracellular cyclic AMP phosphodiesterase in that both are extremely heat stable proteins (Lynch and Farrell, 1985).Plasmodia ofP. flavicomum were grown as previously described by Lynch and Farrell (1984). After reaching stationary phase (about 7 days)the microplasmodia were separated from the medium by centrifugation. The medium was either used immediately or stored frozen at -20°C.Cyclic GMP phosphodiesterase activity was determined by an anion exchange resin procedure using JH-cyclic GMP as the substrate (Lynch andCheung, 1975). Allvalues were corrected for binding of 3H-guanosine to the AGI-X2resin. Cyclic GMP phosphodiesterase activity was also deter-mined by descending paper chromatography using a solvent system of1M ammonium acetate and 95% ethanol (15/35, v/v). Spots correspondingto (3H)-5 'GMP were visualized under ultraviolet light, cut out and the radioactivity determined.Extracellular cyclic GMP phosphodiesterase activity was determined in crude medium and also in partially purified fractions. The enzymewas purified from spent medium by ammonium sulfate fractionation. Medium (100 ml) was adjusted to 30% ammonium sulfate, centrifuged at48,000 xg for 30 min and the pellet discarded. The supernatant fraction was raised to 80% ammonium sulfate and centrifuged at 48,000 xg for30 min. This 30-80% pellet was collected and resuspended in a small volume of20 mM Tris HC1, pH7.6 and dialyzed overnight against the same buffer.140Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42http://scholarworks.uark.edu/jaas/vol39/iss1/42Arkansas Academy of Science Proceedings, Vol. XXXIX,1985 141Arkansas Academy of ScienceThe activity of phosphodiesterase in the starting medium and in a partially purified ammonium sulfate pellet is shown in Table 1.The sametable also shows that this enzyme is heat stable, retaining up to 84% of the unheated activity after exposure to a boiling water bath for 5 min.Extracellular cyclic AMPphosphodiesterase from the same organism was previously shown toalso be heat stable (Lynch and Farrell, 1985). Table1 shows heat resistance at 5 min but gives no indication of how long the enzyme can withstand exposure to 100°C. Figure 1 shows cyclic GMPphosphodiesterase activity remaining after heating for up to twenty minutes in a boiling water bath. Both the starting medium and the ammoniumsulfate pellet retained almost full phosphodiesterase activity even after twenty minutes of exposure to heat.The effect of various compounds on partially purified cyclic GMP phosphodiesterase activity is shown in Table 2. MIX, theophylline andcaffeine are all competitive inhibitors of phosphodiesterase. All three compounds inhibited enzyme activity withMIXbeing the most potent. Chlor-promazine (a phenothiazine) and Compound 48/80 (a condensation product of N-methyl-p-methoxy-phenethylamine with formaldehyde) are thoughtto inhibit calmodulin dependent phosphodiesterase by binding to calmodulin (Levin and Weiss, 1976; Gietzen et al., 1983). Both of these com-pounds stimulated plasmodial cyclic GMP phosphodiesterase. The mode of action of these two compounds on this enzyme is unknown.Table 3 shows cyclic GMP phosphodiesterase activity at various pH values, a pH of 7.0 showed maximum enzyme activity with markedlyreduced activity at pH4.0 and 5.0. This is similar to cyclic AMP phosphodiesterase from the same organism in that the optimum pH for enzymeactivity does not coincide with the optimum pH for growth. Uninoculated growth medium had a pHof 4.3 and after inoculation and growth,the pHof the medium rose to between 5 and 6. Under the growth conditions, cyclic GMP phosphodiesterase should be relatively inactive duringthe early stages of growth (the first few days after inoculation). Then, as the pH increases and approaches 5.0, the enzyme should become increas-ingly active.All the previous enzyme assays were done using the resin assay which measures 3H-guanosine as the end product. To insure the validity ofour heat stability data, an alternate procedure for measuring phosphodiesterase activity was utilized. This procedure involved separating the pro-duct of phosphodiesterase (3H-5 '-GMP) by paper chromatography. Figure 2 shows a continuous assay of cyclic GMP phosphodiesterase for bothheated and unheated samples as measured by paper chromatography. As can be seen, the heated sample retained enzymatic activity and productaccumulation was linear with time.As mentioned previously, extracellular cyclic AMPphosphodiesterase from the same organism is also heat stable. Work is presently under-way in our laboratory to determine ifthe cyclic AMPenzyme is a di tintt protein from the cyclic GMP phosphodiesterase reported here and whetherthe two enzymes are indeed one and the same protein.ACKNOWLEDGMENTSWe would like to thank Mrs. Ann Durgun for excellent clerical assistance. This research was supported by NSF EPSCORGrant #ISP-801 1447, by an Altheimer Foundation Grant and by the College ofSciences ORST (Office of Research in Scienceand Technology).LITERATURE CITEDGIETZEN, K., E. SANCHEZ-DELGADO, and H. BADER. 1983.Compound 48/80: Apowerful and specific inhibitor ofcalmodulin-dependent ca2 + -transport ATPase. IRCS Medical Sci. 11:12-13.LYNCH,T. J., and M. E. FARRELL. 1984. Comparison of cAMPphosphodiesterase from the haploid and diploid stages of the myx-omycete Physarum flavicomum. Cytobios. 39:71-79.LEVIN, R. M., and B. WEISS. 1976. Mechanism by which psycho-tropic drugs inhibit adenosine cyclic 3 ' 5 '-monophosphatephosphodiesterase of brain. Mol. Pharmacol. 12:581-589.LYNCH, T. J., and M. E. FARRELL. 1984. Extracellular phos-phodiesterase from the growth medium of the tnyxomycetePhysarum flavicomum. Proc. Ark. Acad. Sci. 38 p. 60-63.LYNCH, T. J., and W. Y.CHEUNG. 1975. Underestimation ofcyclic3 ',5 '-nucleotide phosphodiesterase by a radioisotopic assay usingan anion-exchange resin. Anal. Biochem. 67:130-138.THOMAS J. LYNCH and MARKS. ROSS, Department of Biology, University of Arkansas at Little Rock, LittleRock, AR 72204.NOTES ON THREE PALUSTRINE NATURAL COMMUNITY TYPES INTHE ARKANSAS OZARKSWithin the last five years three distinct palustrine (sensu Cowardin et al., 1979) community types not previously described in the literatureas occurring in Arkansas have been recognized by the authors. The two fen community types have been reported previously in the Ozark Plateausofsoutheastern Missouri (Orzell, 1983). Each of the recognized community types occur within the Ozark Natural Division(Pell, 1983) and is largelyrestricted to that part of the state. This paper represents an attempt to describe and classify these little-known natural communities.The basis forour classification is the repeated occurrence on the landscape ofparticular combinations ofvegetation structure and physiognomy,soil moisture regime, soil reaction, geologic substrate, topographic position, and species composition. Species composition, though obviously im-portant, is not an overriding concern; in fact, examples of a given "natural community type" may vary considerably in this respect. A naturalcommunity classification emphasizes consistent ecological differences, not those due to the vagaries of dispersal or disturbance history. Inmanycases, more traditionally defined "plant community types" may be recognized within each natural community type.The natural community descriptions presented here include information on distribution, topography, soils, community size and structure,natural processes, status and threats, and dominant and characteristic plant species. Dominant plants are species consistently occurring as majorvegetation components (relative cover > 25 percent, based on visual estimates) of the type, though they need not dominant every example of thattype. Characteristic plants are those typical or representative species that provide a reliable indicator of a given type. Dominant and characteristicspecies are not necessarily restricted to the types but, when considered together with other natural features, are diagnostic. Characteristic specieswere identified by constructing synthesis tables from species checklists compiled during field surveys (Mueller-Dombois and Ellenberg 1974). Four-141Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42Published by Arkansas Academy of Science, 1985

Demonstration of a Heat-Stable Cyclic GMP Phosphodiesterase in the Medium of Physarum flavicomum

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 39 Article 42 1985 Demonstration of a Heat-Stable Cyclic GMP Phosphodiesterase in the Medium of Physarum flavicomum Thomas J. Lynch University of Arkansas at Little Rock Mark S. Ross University of Arkansas at Little Rock Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Biochemistry Commons Recommended Citation Lynch, Thomas J. and Ross, Mark S. (1985) "Demonstration of a Heat-Stable Cyclic GMP Phosphodiesterase in the Medium of Physarum flavicomum," Journal of the Arkansas Academy of Science: Vol. 39 , Article 42. Available at: https://scholarworks.uark.edu/jaas/vol39/iss1/42 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. Arkansas Academy of Science Proceedings, Vol. XXXIX,1985 139Arkansas Academy of ScienceTable 2. Skeletal characteristics and standardized discriminant func-tion coefficients that maximize the discrimination among 68 male andfemale Black-capped and Carolina Chickadees from the Central Plainsand Ozark Plateaus regions.Discriminant functionFirst SeconSkeletal characteristic d:h 0.75 -0.25igth 0.42 -0.36I -1.02 0.81irpus length 0.25 0.46i 0.15 0.68ridth 0.47 -0.40Femur lengthHumerus lenUlna lengthCarpometacarpusFIRST DISCRIMINANTFUNCTION Keel lengthFigure. Ordination with respect to the first two discriminant functionsof 68 skeletal specimens ofBlack-capped and Carolina Chickadees.(Darkened symbols represent Black-capped Chickadees, open onesCarolina Chickadees. Squares are male birds, circles, females. Arrowsindicate the specimens in question.)Synsacrum widtin the smallest group. Because we had only 14 female Black-capped and Carolina Chickadees, it was necessary to a priorireduce the number ofvariables used to 13. Only the 15 variables that showed univariate significance were considered (Table 1), and from these culmen depth and scapulalength were omitted (the former because it is difficult to measure accurately, the latter because of great variation within groups). The 13 variableswere used in a step-wise DFA, using the criterion ofmaximizing the Mahalonobis distance among the 4 groups (males and females of both Black-capped and Carolina Chickadees). The infrequent missing values were estimated using the mean values for the appropriate group (e.g., Carolinafemale). The whole analysis was repeated using 75 chickadees by adding specimens from a wider geographical range. The results of the two analyseswere essentially similar to those described below, which are based on the one using the 68 specimens.The step-wise DFA identified 6 variables that maximized the separation among the 4groups of chickadees. These variables, and their standar-dized discriminant functions coefficients are listed in Table 2. As can be seen from the Figure, the two species are separated along the first discrimi-nant function axis (DF-1), accounting for 82.4% of the variance among the four groups.Ulna length is the variable that contributes most to the separation between species with femur length and synsacrum width being of secondaryand tertiary importance (Table 2). In total, Black-capped Chickadees, which have relatively large positive values on DF-1, have a combinationof a relatively short ulna, long femur and humerus, and wide synsacrum. The measures of wing length, other than ulna length, interestingly arepositively correlated to DF-1. The second DF (accounting for an additional 17.4% of the variation among the groups) separates male and femaleBlack-capped Chickadees but not the sexes of Carolina Chickadees (Figure). Ulna length, again, is the most important variable on the axis, withmale atricapillus having longer ulnae and keels, but relatively narrower synsacra and shorter humerae than females (Table 2).Inevaluating the precision of the discriminat function analysis, 82% of the specimens (56 out of 68) were correctly categorized as to groupaffinity. Eleven of the misidentifications were withregard to sex within species, and only one specimen was grouped with the wrong species. Thiswas the female Black-capped Chickadee from Phillips Co., Kansas (KU 61722 9 )> tnat is clustered with the Carolina Chickadees in the Figure.The analysis identified it as a female Carolina Chickadee. Thus 99% of the specimens were correctly positioned with respect to species.The two specimens in the Kansas collection that were suspect as toidentification were both identified by the analysis as being Carolina Chickadees(marked by the two arrows in the Figure). As is shown in the Figure, the male specimen clearly is a Carolina Chickadee whereas the female specimenis close to the misclassified Black-capped Chickadee. Infact this female bird was identified with a rather high probability ofbeing a Carolina Chickadee.Ithad a 69% probability ofbeing a female Carolina (which it is), and 26% probability of being a male Carolina, which leaves only a 5% probabilityof being a Black-capped Chickadee.Insummary, Black-capped and Carolina Chickadees are readily identified from skeletal material and two suspect skeletal specimens labeledBlack-capped Chickadees from Arkansas were indeed Carolina Chickadees. Thus, there are still no records of the Black-capped Chickadee in Arkansas.(In completing this study, Rising's contribution was supported by the Natural Sciences and Engineering Research Council of Canada. JimDick of the Royal Ontario Museum, and Marion Mengel and Steven Johnson, of the University of Kansas Museum of Natural History, were veryhelpful inmaking specimens available. Thanks are due Janet Mannone for managing the data. J. D. Black was especially helpful for his comments.)DOUGLASJAMES, Department of Zoology, University ofArkansas, Fayetteville, AR 72701, and JAMES D.RISING, Department of Zoology,University of Toronto, Toronto, Ontario M53 IA1, Canada.DEMONSTRATION OF A HEAT-STABLE CYCLIC GMP PHOSPHODIESTERASEINTHE MEDIUMOF PHYSARUM FLA VICOMUMAlthough the role ofcyclic AMPin cellular regulation has been well characterized during the last two decades, the function ofcyclic guanosinemonophosphate (cyclic GMP) remains vague. Cyclic GMP phosphodiesterase is the enzyme responsible fordegrading cyclic GMP to 5 'GMP andthus is of major importance in maintaining cellular levels of cyclic GMP. We have identified an extracellular cyclic GMP phosphodiesterase in139Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42Published by Arkansas Academy of Science, 1985Arkansas Academy of Science Proceedings, Vol. XXXIX,1985140General NotesTable 1. Effect of Heat on Cyclic GMP Phosphodiesterase Activity.nMole/min/ml % ActivityUnheated Heated RemainingStarting Medium 146 86 60%30-80% Pellet 160 134 84%Samples of starting medium (sm) and ammonium sulfate 30-80% pelletwere assayed for cGMP phosphodiesterase activity without heating(unheated) and after a 5 minexposure to a boiling water bath (heated).The sm is an average of four experiments and the 30-80% pellet is anaverage of three experiments.Table 2. Effect of Various Compounds on Cyclic GMPPhosphodiesterase Activity.Additive Concentration ijMole/min/ml % ActivityControl 0 162 100%MIX 7 MM 50 31%Theophylline 10 MM 63 39%Caffeine 10 MM 94 58%Chlorpromazine 500 pM 207 128%Chlorpromazine 1 MM 207 128%Compound 48/80 10 pG 253 156%Compound 48/80 100 jjg 225 139%Dialysed samples ofthe ammonium sulfate 30-80% pellet were assayedin the presence of the above indicated reagents. MIXis 3-isobutyl-l-methylxanthine.Table 3. Effect of pH on cGMP Phosphodiesterase Activity.Starting Medium 30-80% pelletpH nMole/m in/ml % Activity nFjole/m in/ml % Activity11% 56 712<4157bl XZ5 7%Z 659 79%6 125100% 830 100%7 22!;<m 1678 20%93Cyclic GMP phosphodiesterase was determined at the pH values in-dicated. A citrate buffer was used for pH 4 and 5, Pipes buffer forpH 6 and 7, and Tris for pH 8.0.Figure 1. Samples of starting medium (SM) and 30-80% ammoniumsulfate pellets were assayed for cyclic GMP phosphodiesterase activityafter being heated in a boiling water bath for the times indicated onthe abscissa.ASSAY TIME (min)Figure 2. Cyclic GMP phosphodiesterase activity inheated and unheatedsamples was measured by paper chromatography. The 30-80% am-monium sulfate pellet for heat treatment was placed in a boiling waterbath for 10 min and then returned to 30 °C. Reaction mixtures of 300fi\ each were prepared for heat-treated and non-heat treated samples.Duplicate 0.025 ml samples were withdrawn at the indicated times andadded to tubes containing 0.005 ml of 2 MHC1 to stop the reaction.Cold 5'GMP (0.005 ml of 20 mM solution) was added and aliquotswere spotted on Whatman filter paper. Descending paperchromatography was done overnight and the 3H-5'GMP spots cut outand counted.the medium from the plasmodium of the myxomycete Physarum flavicomum. This cyclic GMP phosphodiesterase appears to be very similar tothe previously reported extracellular cyclic AMP phosphodiesterase in that both are extremely heat stable proteins (Lynch and Farrell, 1985).Plasmodia ofP. flavicomum were grown as previously described by Lynch and Farrell (1984). After reaching stationary phase (about 7 days)the microplasmodia were separated from the medium by centrifugation. The medium was either used immediately or stored frozen at -20°C.Cyclic GMP phosphodiesterase activity was determined by an anion exchange resin procedure using JH-cyclic GMP as the substrate (Lynch andCheung, 1975). Allvalues were corrected for binding of 3H-guanosine to the AGI-X2resin. Cyclic GMP phosphodiesterase activity was also deter-mined by descending paper chromatography using a solvent system of1M ammonium acetate and 95% ethanol (15/35, v/v). Spots correspondingto (3H)-5 'GMP were visualized under ultraviolet light, cut out and the radioactivity determined.Extracellular cyclic GMP phosphodiesterase activity was determined in crude medium and also in partially purified fractions. The enzymewas purified from spent medium by ammonium sulfate fractionation. Medium (100 ml) was adjusted to 30% ammonium sulfate, centrifuged at48,000 xg for 30 min and the pellet discarded. The supernatant fraction was raised to 80% ammonium sulfate and centrifuged at 48,000 xg for30 min. This 30-80% pellet was collected and resuspended in a small volume of20 mM Tris HC1, pH7.6 and dialyzed overnight against the same buffer.140Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42https://scholarworks.uark.edu/jaas/vol39/iss1/42Arkansas Academy of Science Proceedings, Vol. XXXIX,1985 141Arkansas Academy of ScienceThe activity of phosphodiesterase in the starting medium and in a partially purified ammonium sulfate pellet is shown in Table 1.The sametable also shows that this enzyme is heat stable, retaining up to 84% of the unheated activity after exposure to a boiling water bath for 5 min.Extracellular cyclic AMPphosphodiesterase from the same organism was previously shown toalso be heat stable (Lynch and Farrell, 1985). Table1 shows heat resistance at 5 min but gives no indication of how long the enzyme can withstand exposure to 100°C. Figure 1 shows cyclic GMPphosphodiesterase activity remaining after heating for up to twenty minutes in a boiling water bath. Both the starting medium and the ammoniumsulfate pellet retained almost full phosphodiesterase activity even after twenty minutes of exposure to heat.The effect of various compounds on partially purified cyclic GMP phosphodiesterase activity is shown in Table 2. MIX, theophylline andcaffeine are all competitive inhibitors of phosphodiesterase. All three compounds inhibited enzyme activity withMIXbeing the most potent. Chlor-promazine (a phenothiazine) and Compound 48/80 (a condensation product of N-methyl-p-methoxy-phenethylamine with formaldehyde) are thoughtto inhibit calmodulin dependent phosphodiesterase by binding to calmodulin (Levin and Weiss, 1976; Gietzen et al., 1983). Both of these com-pounds stimulated plasmodial cyclic GMP phosphodiesterase. The mode of action of these two compounds on this enzyme is unknown.Table 3 shows cyclic GMP phosphodiesterase activity at various pH values, a pH of 7.0 showed maximum enzyme activity with markedlyreduced activity at pH4.0 and 5.0. This is similar to cyclic AMP phosphodiesterase from the same organism in that the optimum pH for enzymeactivity does not coincide with the optimum pH for growth. Uninoculated growth medium had a pHof 4.3 and after inoculation and growth,the pHof the medium rose to between 5 and 6. Under the growth conditions, cyclic GMP phosphodiesterase should be relatively inactive duringthe early stages of growth (the first few days after inoculation). Then, as the pH increases and approaches 5.0, the enzyme should become increas-ingly active.All the previous enzyme assays were done using the resin assay which measures 3H-guanosine as the end product. To insure the validity ofour heat stability data, an alternate procedure for measuring phosphodiesterase activity was utilized. This procedure involved separating the pro-duct of phosphodiesterase (3H-5 '-GMP) by paper chromatography. Figure 2 shows a continuous assay of cyclic GMP phosphodiesterase for bothheated and unheated samples as measured by paper chromatography. As can be seen, the heated sample retained enzymatic activity and productaccumulation was linear with time.As mentioned previously, extracellular cyclic AMPphosphodiesterase from the same organism is also heat stable. Work is presently under-way in our laboratory to determine ifthe cyclic AMPenzyme is a di tintt protein from the cyclic GMP phosphodiesterase reported here and whetherthe two enzymes are indeed one and the same protein.ACKNOWLEDGMENTSWe would like to thank Mrs. Ann Durgun for excellent clerical assistance. This research was supported by NSF EPSCORGrant #ISP-801 1447, by an Altheimer Foundation Grant and by the College ofSciences ORST (Office of Research in Scienceand Technology).LITERATURE CITEDGIETZEN, K., E. SANCHEZ-DELGADO, and H. BADER. 1983.Compound 48/80: Apowerful and specific inhibitor ofcalmodulin-dependent ca2 + -transport ATPase. IRCS Medical Sci. 11:12-13.LYNCH,T. J., and M. E. FARRELL. 1984. Comparison of cAMPphosphodiesterase from the haploid and diploid stages of the myx-omycete Physarum flavicomum. Cytobios. 39:71-79.LEVIN, R. M., and B. WEISS. 1976. Mechanism by which psycho-tropic drugs inhibit adenosine cyclic 3'5 '-monophosphatephosphodiesterase of brain. Mol. Pharmacol. 12:581-589.LYNCH, T. J., and M. E. FARRELL. 1984. Extracellular phos-phodiesterase from the growth medium of the tnyxomycetePhysarum flavicomum. Proc. Ark. Acad. Sci. 38 p. 60-63.LYNCH, T. J., and W. Y.CHEUNG. 1975. Underestimation ofcyclic3 ',5 '-nucleotide phosphodiesterase by a radioisotopic assay usingan anion-exchange resin. Anal. Biochem. 67:130-138.THOMAS J. LYNCH and MARKS. ROSS, Department of Biology, University of Arkansas at Little Rock, LittleRock, AR 72204.NOTES ON THREE PALUSTRINE NATURAL COMMUNITY TYPES INTHE ARKANSAS OZARKSWithin the last five years three distinct palustrine (sensu Cowardin et al., 1979) community types not previously described in the literatureas occurring in Arkansas have been recognized by the authors. The two fen community types have been reported previously in the Ozark Plateausofsoutheastern Missouri (Orzell, 1983). Each of the recognized community types occur within the Ozark Natural Division(Pell, 1983) and is largelyrestricted to that part of the state. This paper represents an attempt to describe and classify these little-known natural communities.The basis forour classification is the repeated occurrence on the landscape ofparticular combinations ofvegetation structure and physiognomy,soil moisture regime, soil reaction, geologic substrate, topographic position, and species composition. Species composition, though obviously im-portant, is not an overriding concern; in fact, examples of a given "natural community type" may vary considerably in this respect. A naturalcommunity classification emphasizes consistent ecological differences, not those due to the vagaries of dispersal or disturbance history. Inmanycases, more traditionally defined "plant community types" may be recognized within each natural community type.The natural community descriptions presented here include information on distribution, topography, soils, community size and structure,natural processes, status and threats, and dominant and characteristic plant species. Dominant plants are species consistently occurring as majorvegetation components (relative cover > 25 percent, based on visual estimates) of the type, though they need not dominant every example of thattype. Characteristic plants are those typical or representative species that provide a reliable indicator of a given type. Dominant and characteristicspecies are not necessarily restricted to the types but, when considered together with other natural features, are diagnostic. Characteristic specieswere identified by constructing synthesis tables from species checklists compiled during field surveys (Mueller-Dombois and Ellenberg 1974). Four-141Journal of the Arkansas Academy of Science, Vol. 39 [1985], Art. 42Published by Arkansas Academy of Science, 1985

https://scholarworks.uark.edu/cgi/viewcontent.cgi?article=2473&amp;context=jaas

Demonstration of a Heat-Stable Cyclic GMP Phosphodiesterase in the Medium of Physarum flavicomum

Abstract

Similar works

Full text

Available Versions

ScholarWorks@UARK

UARK (University of Arkansas )