Flavell, R.

Fungal Genetics Reports

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Kansas State University

Fungal Genetics Reports Volume 14 Article 33 Comments on enzyme assays R. Flavell Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Flavell, R. (1969) "Comments on enzyme assays," Fungal Genetics Reports: Vol. 14, Article 33. https://doi.org/10.4148/1941-4765.2064 This Enzyme Methodology is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Comments on enzyme assays Abstract Comments on enzyme assays This enzyme methodology is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol14/iss1/33 Wild ‘ype Neurosporo  crosso  (74-OR-A) woz  grown on 196  lactose for 5 days and the mycelio were filtered out on o  &hoer-funnel. The mycelio were extracted  with 10 ml of 0.01 M No phosphate, pH 7.5, per g of wet weight. The mycelio were home-genized  in on Omni-Mixer, sonicoted, and then stirred for 2 hrs ot 4°C. After centrifugotion  at 20,000 x g for 20 min,  the super-noton+ war used os  crude extract. The growth medium after  filtration  wm  concentrated  by dialysis  ogointt  dry sucro~c. Samplerfor electrophoresis  contained z 0.25 mg protein.Crude extract  gave reoctiom  with ONPG ot three distinct sites on the gel, The 7.5 enzyme hod on Rf of 0.046, the 4.2 en-zyme hod on Rf of 0.250, ond o third form of the enzyme hod on Rf of 0. 150. When the growth medium was  electrophoresed,  oc-tivity  oppeored  ot either one or two sites, depending upon  the age  of the culture. Medium from o young culture shaved only thenew form of the enzyme (pH  4.5) w I e  medium from on old culture contained both the 4.2 and 4.5 forms with o  predominanceh.1of the former. Between these two extremes there were gradations  in the proportions of the two forms. This work supported in portby the NIH  Training Grant  in Genetics (TOI-GMOl316)  to Florida State University. - - - Genetics Laboratories, Department ofBiological  Science, Florida State University, Tollohossee,  Florida 32306.Morgan, D. H. The msay of orginose. Many  methods of orginore  ossoy  in voriws organisms  hove been published. Thefoilwing  procedure has  been found to tiork  well with crude extracts  of Neuraporo.Frozen myceliol  pods are ground in o  chilled mortar with gloss powder and 5-10 times their weight of pH 7.0 0.025 M moleic acid/sodium hydroxide buffer  containing  mongonese  chloride (0.005  M) and  dithioerythritol (0.002 M). An -SH  reagent  is possiblysuperfluous in undiolyned  extracts  but has  been found to stobilire  the enzyme during dialysis.A reaction  mixture cdnsists  simply of 0.2 m! of enzyme ond 0.3 ml of 0.3 M orginine. The orginine solution is adjusted  to pH9.5 and final  PH in the mix is &out  9.2, the orginine itself prov’oviding  adequate buffering. Incubation  is ot  37”C,  the enzyme be-ing po-incubated  ot this temperature  for IO-15 min before arginine  addition. The reaction  is stopped with 4.5 ml of 2% TCA,zero-time blanks being stopped before orginine addition. Ornithine  estimation  (see below ) is carried  out on 0.5 ml samples ofthe stopped reaction  mix. (Wh en it is  desired to use lower sutstrote  concentrations  ond therefore to detect lower levels of orni-thin=,  to stay in the linear region dilution with TCA  is reduced or avoided oltcgether  by stopping with the acid ninhydrin reagentused for the ornithine estimation.)The estimation  of ornithine in assaying for orginare  or acetyl-ornithine/glutclmate  tronsacetylose. The method of ornithineestimation  used by Vogel and Banner ( 1956 J. Riol.  them. 218:97)  for the ossoy of ocetylornithinose  is also applicable  to thearray of arginase  and  ocetylornithine/glutomo+e  tronsacetylose. It is quicker than the commonly-used method of Chinord (1952J. Biol. Chem.  199: 91  ) ond the ninhydrin mix used (mode up in 0.4 hl citric acid and methyl cellosolve)  is pleosonter  to deolwith than that  of Chinord (6 M phosphoric acid ond glacial  acetic acid). The optimum boiling time for arginose  samples is 25min. Both glutornate  and orginine give rise to a deep blue color after  the final  addition  of NoOH. This  persish  until the samplesore subjected to vigorous Vortex mixing (30-60 set  ) when it disoppeors, revealing the stable golden-brown color which is readot 470 mp. It is necessary to read  ogoinst  no-ornithine blanks containing  appropriate quantities  of orginine or glutamate,  both ofwhich give appreciable  blank values.  Sensitivity is obcut three-fold lower than with Chinard - 0.4 pmoles of ornithine per ODunit ot 470 mp  in the presence of 9 pmoles orginine per sample. The reaction  is lineor  ot least up to 0.5 pmoles ornithine perSOfllpl@.This work was supported by the Gceney  Fund, Colifornio  Institute of Technology, The hospitality  and encouragement  ofN. H. Horowitz was greatly  appreciated. - - - John Inner Institute, Colney Lone, Norwich NOR 7OF. England.Flovell,  R. Comments on arsoy methods. The following ossoyr hove been used for Neurosporo  os  reported by Flovell  andFinchom  (1968 J. Eocteriol.  95: 1063). Any modifications  used since then orenoted after the relevant  osroy. The original  references to the ossoys  ore given inthe paper  mentioned above.lsocitrote  Iyore, E.C. 4. I .3. I.;  but with I5 pmoles  of DL-isocitrote.Molote  synthase,  E.C. 4. I  .3.2.;  but with 0.04 pmoles of Acetyl C oA.Phcsphoenolpyruvote  corboxykinase,  E.C 4. I.  1.32.; but with 9 pmoles phosphoenolpyruvote.Citrotesynthore,  E.C. 4.1.3.7.;  butwith  0.04 pmoles of Acetyl  CoA.Acetyl  CoA synthetose  E.C. 6.2. I.  I.Molote  dehydrogenose,  E.C. I.  I.  1.37.NADP-l inked lsocitrote  dehydrogenose,  E.C.  I. I. 1 .42.NAD-linked lsocitrote  dehydrcgenase,  E.C. I.  I.  1.41.; but with 8 mM DL-isoctote and I mM NAD.Fumorote  hydrotose,  E.C. 4.2. I.  2.The following assays have also been  used without modification of the method cited in the reference following each enzyme:Succinote  dehydrogenase,  E . C .  1.3.99.  I.  (King 1963 J .  Biol.  Chem. 238:4032)ATP  citrate  lyase,  E.C.  4 .  1.3.6.  (Atk inson Biochem. Sot.  Symp. 27:31  ).- - - Department of Biological Sciences, Sfonford  University, Stanford,  Colifornio  94305.

Comments on enzyme assays

R. Flavell

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Comments on enzyme assays

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