Effects of some glycosidases and of periodate on the activity of the glycoprotein NAD ( P )

Effects of glycosidases and periodate on glycoprotein NAD(P)ase Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This research note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol23/iss1/5 eighth conidiotion bands. The tuber were later scanned with CI densitometer coupled to CI chart recorder. From these tracings and the previous growth front measurements, the elapsed time between the middle of the third and eighth conidiotion bonds was calculated for each growth tube. If this interval for on irradiated tube was shorter or longer than the average interval from a set of unirmdioted control tubes, then the difference was recorded os CI phase advance or delay respectively. Figure I shows the combined doto from nine independent experimentr. OS I* ~o*Icm.Iow IAND &se shifts ore plottedasadvonceordelayin fractionsof acycle versus the stage ot which the culture was irradiated. It is clear thot maxinwm 0.4 Phase odvonces occurred when ultraviolet light was administered mound ~ i 1-1 oo3(z the middle of the fourth conidiation bond. Lesser odvonces occurred when the light pulses fell during the early ond late phases of conidiation. In 00 00 0 0 general, only small phase shifts resulted when the tubes were irradiated ” *Itoo0 O” 0 0 Lo0 D during the nonconidioted or interband phases of growth. A very few ini stances of substontiol phase delay were observed when irrodiotion occurr0 a” 01 ed during late interbond. These results ore partially in accord with the 5 0 : pattern of phase shifts thot Sargent ond Briggs induced with white light. f p 0~1 They also found maximum phase advances when white light exposures coi 0~3 incided with the middle of o conidiotion bond. However, they did ob01 serve substantial phase delclys with white light pulses administered throughout the late interband and early conidiotion phases of growth. 0~1’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ t 0.4 05 06 0.7 01 07 IO I/ I2 I~3 I. /, I~6 The effectiveness of short wavelength ultraviolet light could indicate was 01 “LlllYlDLEl 111101.110” IN CICLSI A,,*, Mm” or ,*110 cowIDI.rIoN S.ND o role for nucleic acids in the phase shifting mechanism. In this context it would be of interest to determine whether phase shifts induced by ultraviolet light could be reversed by visible light. However, o rigorous test of this possibility requires that there be phases in the cycle where ultraviolet light causes substantial phase shift while visible light does not. The rather close correspondence in the ultraviolet ond white light induced phase shift profiles makes this test very difficult,if not impossible,to carry out. Biology Division, Kansas State University, Manhattan, Konsos 66506. Urey, J. C. and D. B. Smith. Effects of some glycosidoses and Everse et 01. (1975 Arch. Biochem. Biophys. 169: 702) ex?tended thar earlier report that Neurospora NAD(P)ose (E.C.3. of periodate on the activity of the glycoprotein NAD(P)ose. 2.2.5)isoglycoprotein contoiningBO% corbohydratebyweight. Field et (II. (1973 Abst. Am. Sot. Microbial. Mtg.) reported that the enzyme reacts specifically with Concanavalin A demonstrating a-monnore or ~-glucose (IS CI terminal residue in the carbohydrate portion. We report here the effects of several glycosidoses and of periodate oxidation upon the enzymic activity of NAD(P)ose. Strain 74-01(23-IA wcls grown on solid Vogel’s medium N plus 2% sucrose to obtain conidia. Enzyme was obtained by washing the conidia with 0.1 M sodium phosphate buffer pH7.5 and removing the conidia by centrifugotion. Enzyme was also extracted from cultures grown on zinc-deficient Fries minimal medium (Koplon et al. 1951 J. Biol. Chem. 188: 397). After harvest, themycelium was homogenized in phosphate buffer pH7.5 and the debris removed by centrifugotion. These different crude enzyme preporations gave indistinguishable results. NAD(P) ose activity was orsayed by the cyanide-addition method of Kaplan et al. (1951). Ficin U-galactosidase (generously provided by Dr. Su-then Li, Tulane University) was incubated with NAD(P)ase ot 25OC in 0.5 M rodiurn acetate buffer pH4.5 for up to I8 hours. Jack Bean a-mannosidase (gift of Dr. Li) was incubated with NAD(P)ose at 25O C in 0.05M acetote buffer pH4.5 for up to 34 hours. E.coli B-goloctosidase was incubated with NAD(P)ose ot 3PC in 0.1 M sodium phosphate buffer pH 7.0 plus 0.1 M mercaptoeth&~ mM MgS04 and 0.2mM MnSO4 for up to 37 hours. Rhizopus sp. a-glucoamylase (Sigma) was incubated with NAD(P) ose at 37oC in 0.1 M acetote buffer pH4.5 plus 1 mM phenylmethyln$ fluoride (PMSF) for up to 14.5 hours. B.subtilis a-amylase (Sigma) was incubated with NAD(P) ose at 25’C in 0.02 M phosphate buffer pH 6.9 plus I mM PMSF for up to 14.5 hours. In every experiment with each of these five glycosidases, no effect of the glycosidose on the activity of NAD(P)ose was detected. Since the NAD(P)ose was not pure, we were “noble to determine whether any sugar residues hod been released from the enzyme. We report in Table I data showing that PMSF does not inhibit NAD(P)ose suggesting that none of the eight swine residues in the enzyme is important for its activity. In the absence of PMSF, the proteinores in the Rhizopus and B.subtilis enzymes rapidly destroyed the NAD(P)ose. Periodate specifically oxidizes diglycols and ominoglycols and is used in glycoprotein analysis (e.g., Spiro 1964 J. Biol. Chem. 239: 567).We performed the oxidations a+ both pH4.0 and 7.5. At pH4.0 NAD(P) ose was incubated ot 25’C in the dark with 0.025M sodium metoperiodote in 0.1 M sodium ocetote buffer. At pH 7.5 NAD(P)ose was incubated ot 25OC in the dork with 0.0125M potassium periodate in 0.1 M Tris buffer. In both cases, the oxidation was stopped by mixing a 0.1 ml oliquot withO.3ml of 0.1 M sodium phosphate buffer pH 7.5 containing 0. I M ethylene glycol. Then NAD(P)ose was assayed by adding 0.1 ml NAD &mg/ml) os in Kaplan’s standard assay. The results in Table II show that periodate rapidly inactivated NAD(P)ose at both pH’s. These results ore consistent with the possibility that the carbohydrate pation of NAD(P) o r e is required for its activity. The greater sensitivity ot the higher pH is consistent with the aminosugars being more important than simple sugars; however, in view of the small mnount of aminosugars in NAD(P) ase and the impurity of our enzyme preparation, this conclusion is tentative. These studies hove been terminated.

In every experiment with each of these five glycosidases, no effect of the glycosidose on the activity of NAD(P)ose was detected.
Since the NAD(P)ose was not pure, we were "noble to determine whether any sugar residues hod been released from the enzyme.We report in Table I data II show that periodate rapidly inactivated NAD(P)ose at both pH's.
These results ore consistent with the possibility that the carbohydrate pation of NAD(P) ore is required for its activity.The greater sensitivity ot the higher pH is consistent with the aminosugars being more important than simple sugars; however, in view of the small mnount of aminosugars in NAD(P) ase and the impurity of our enzyme preparation, this conclusion is tentative.These studies hove been terminated.
Table I Table 11 Lock monnore or ~-glucose (IS CI terminal residue in the carbohydrate portion.We report here the effects of several glycosidoses and of periodate oxidation upon the enzymic activity of NAD(P)ose.Strain 74-01(23-IA wcls grown on solid Vogel's medium N plus 2% sucrose to obtain conidia.Enzyme was obtained by washing the conidia with 0.1 M sodium phosphate buffer pH7.5 and removing the conidia by centrifugotion.Enzyme was also extracted from cultures grown on zinc-deficient Fries minimal medium (Koplon et al. 1951 J. Biol.Chem.188: 397).After harvest, themycelium was homogenized in phosphate buffer pH7.5 and the debris removed by centrifugotion.These different crude enzyme preporations gave indistinguishable results.NAD(P) ose activity was orsayed by the cyanide-addition method of Kaplan et al. (1951).Ficin U-galactosidase (generously provided by Dr. Su-then Li, Tulane University) was incubated with NAD(P)ase ot 25OC in 0.5 M rodiurn acetate buffer pH4.5 for up to I8 hours.Jack Bean a-mannosidase (gift of Dr. Li) was incubated with NAD(P)ose at 25O C in 0.05M acetote buffer pH4.5 for up to 34 hours.E.coli B-goloctosidase was incubated with NAD(P)ose ot 3PC in 0.1 M sodium phosphate buffer pH 7.0 plus 0.1 M mercaptoeth&~ mM MgS04 and 0.2mM MnSO4 for up to 37 hours.Rhizopus sp.a-glucoamylase (Sigma) was incubated with NAD(P) ose at 37oC in 0.1 M acetote buffer pH4.5 plus 1 mM phenylmethyln$ fluoride (PMSF) for up to 14.5 hours.B.subtilis a-amylase (Sigma) was incubated with NAD(P) --ose at 25'C in 0.02 M phosphate buffer pH 6.9 plus I mM PMSF for up to 14.5 hours.

NAD
showing that PMSF does not inhibit NAD(P)ose suggesting that none of the eight swine residues in the enzyme is important for its activity.In the absence of PMSF, the proteinores in the Rhizopus and B.subtilis enzymes rapidly destroyed the performed the oxidations a+ both pH4.0 and 7.5.At pH4.0 NAD(P) ose was incubated ot 25'C in the dark with 0.025M sodium metoperiodote in 0.1 M sodium ocetote buffer.At pH 7.5 NAD(P)ose was incubated ot 25OC in the dork with 0.0125M potassium periodate in 0.1 M Tris buffer.In both cases, the oxidation was stopped by mixing a 0.1 ml oliquot withO.3ml of 0.1 M sodium phosphate buffer pH 7.5 containing 0. I M ethylene glycol.Then NAD(P)ose was assayed by adding 0.1 ml NAD &mg/ml) os in Kaplan's standard assay.The results in Table at 25OC in the dark a+ the pH shown, with or without perkdote.Activity is the absorbance a+ 325nm of NAD-CN in the K&on sodium phosphate buffer pH6.9, with or without I r&l PMSF.Activity is the absorbance ot 325 nm of NAD-CN in the Kaplan orsoy.Average of +wo triols.array.Average of tw., trials.---(Biology Department, Wheaton College, Norton, MA) Current addresses: 118 Porker Street, Attleboro, Marrachurefk 02703 and Running Fox Farm, RFD 4, West Chester, Pennsylwnia 19380.