Assay of proteolytic enzyme(s) from Neurospora crass

Drysdale, R. B.

Fling, M.

Fungal Genetics Reports

English

Kansas State University

Fungal Genetics Reports Volume 8 Article 16 Assay of proteolytic enzyme(s) from Neurospora crassa R. B. Drysdale M. Fling 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 Drysdale, R. B., and M. Fling (1965) "Assay of proteolytic enzyme(s) from Neurospora crassa," Fungal Genetics Reports: Vol. 8, Article 16. https://doi.org/10.4148/1941-4765.2112 This Technical Note 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. Assay of proteolytic enzyme(s) from Neurospora crassa Abstract Assay of proteolytic enzyme(s) from Neurospora crassa This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol8/iss1/16 C. CPK (from Davis 1965 B&him.  Biophys. Acto 107:54).HC03-  +  NH4+  +  A T P Mg++PH  8.2  ’ *’ + ADP’In this ossoy,  CAP is meosured  by including OTC and ornithine in excess in reaction mixtures and measuring the citrullineformed at  the end of the reaction time. For crude extracts of most strains, it may be unnecessary to odd OTC, since it is pre-sent in sufficient activity  for measurement of CPK. If it must be added, it may be purified from arg-3 strains by the method ofDavis  (1965 B&him.  Biophys. Acta  107:44).  Alternatively,  crude extracts of erg-3  may be usxthe OTC has  derepressedsufficiently. The arg-3 strain  is used because it has  no CPK itself.Using a 13  x 100  mm tube, combine, in CI  final volume of 0.5 ml, 50~ moles Tris-HCI (PH 8.4),  8 pmoles M&l  , 30 pmolesNH4CI,  IO pmoles  KHC03, 3 pmoles ATP Nq,  2.5 pmoles L-ornithine  HCI, excess OTC (IO-20 units) and  a rate-7.grnntnngextract (0.5 mg protein for most wild types). As a rule, a substrate mixture is prepared by adding buffer3, MgC12  and ornithine. (The KHC03 should not be exposed to acid pH).  The ATP is then added andthe pH of the mixture adjusted to PH 8.2 (th’II is usually  unnecessary if pH  8.4 Tris buffer is used). This mixture is kept in thecold; just before use, OTC is added. The concentration of ingredients should be such as to contain the requirements of 0 singlereaction mixture in 0.2 ml. The mixture is then distributed to tubes containing experimental extrcxts (0.3 ml, with water) witha ZOO-lambda micropipette, the tubes shaken, and immediately put in a 37’C  water bath. Incubation continues for 30 minutes,after which the reactions are  stopped in the order in which they were  storted  by adding 0. I ml 2M HCl04  with a IOO-lambdamicropipette. The tubes are allowed to stand at room  temperature or  on ice for 5-10 minutes. They are  then centrifuged atlow speed to remove  protein, and 0.4 ml of the supernatant  (z/3  of the acidified reaction mixture) is tested for citrulline inthe Koritz-Cohen test, scaled down by one-half. 0 rice again,  the appropriate control is a time zero  blank.D. Calorimetric  test for carbomyl compounds (After Koritz  and Cohen k.  c;  Crockoert  and  Schram  1958  Bull Sot.Chim. Biol.  40: 1093 :Reagents: 50% (v/v) H2S04;  3% diocetylmonoxime (2,3-butanedione  mcmoxime,  Eastman Organic Chem., Cat. No. 86.Store in darkness.); 0.4% p-diphenylamine sulfonic  acid sodium solt  ( Eastman Organic Chem. Cat. No. 5897. Store in dark-ness. ); I N HCI; 1% K2S20B (Store in cold).Method: Standard, matched Klett-Summerson colorimeter  tubes are used, as the final color determination may  be made di-rectly in them. Samples from control and experimental reaction mixtures (ATC and OTC) ore made up to 2.0 ml if necessary.A calorimetric  blank (water) and  o standard  (0. I pmole  citrulline or 1 .O pmole  US) ore used as controls. All further stepsshould be carried out in the dark, where indicated, cmd  in any case  out of direct, bright light.To each 2 ml sample, 4.5 ml of a mixture made just prior to use (without exposure to bright light) in the proportions of4.0 ml H2SO4,  0.2 ml diacetylmonoxime,  0.2 ml p-diphenylomine sulfonate,  and  0.1 ml HCI is added and stirred thoroughly.This may be accomplished by snapping the tubes, by the use of a Vortex mixer, or by bubbl’ gun air through them with a glasstube connected to on air line. (In the latter case, the (rev&ion  tibe  should  be rinsed between uses). The tubes are  cappedwith capillary stoppers, marbles, or aluminum caps, such that vapors may escape,  but water  cannot enter the tube.The rack of tubes is placed in boiling water  in the dark (a roasting pan  with o cover is convenient) for 10 minutes. Afterthis time, they are removed and cooled in o pan of tap  water for 5 minutes or Y), again in the dark.To each tube, 0.2 ml K2520B  is added  quickly wiih o 0.2 ml serological  pipette aimed at the surface of the fluid in theexperimental tube. The tubes are mixed thoroughly and quickly, and  the tubes ore replaced in bailing water  in the dark forexactly  one minutes. They are then cooled for 5-10 minutes in the dark, and each tube is read against  the reagent blank in aKlett-Summerson calorimeter  with CI  x54  filter (maximum transmission, 540  mp). Tubes should be kept in the dark until #heyare read, and  they should be read  in a short period of time. The readings ore converted to proles  (per tube) by comparisonwith the standard. The pmoles of product per reaction mixture may then be calculated (after subtraction of the time zerocontrol values) with the knowledge of the volume used in the color test 0s  CI  proportion of the volume of the HClOq-treatedreaction mixture. Specific activities are generally expressed as pmoles product per mg protein per hour.In the case of CPK assays, all ingredients of the color test ore reduced by one half, and the readings (and color test, ifpossible) are done in flat-bottomed Klett  tubes having  minimum volumes of 2.5-3.0 ml.Wild type (74A) contains the enzymes in the following range  of specific activities if the mycelium is grown according tothe shake flask method: OTC IO-20 pmoles  citrulline/mg  protein/hour; ATC 2-4 p moles US/mg protein/hour; CPK 0.1-0.2pmole  ci+rulline/mg  protein/hour. - - - Department of Botany, University of Michigan, Ann Arbor, Michigan.Drysdale, R. 8.  and M. Fling. Assay of proteolyticenzyme  s from Neurosporo  crasso.free and peptide,  in the perchloric acid supernatant.Proteolytic activity is measured by incubating samples ofextract with casein,  stopping the reaction by addition ofperchloric acid and measuring the amount of tyrosine,  bathA. Preparation of extract: The mycelium is pressed dry on filter paper and then ground with a little sand in a cold mortar.Sodium phosphate buffer (0.1 a;  pH 6.0; IO parts  v /w)  is added and  the extmct  centrifuged ot 10,000 x g for IO minutes.The supermtont  is decanted and used for the enzyme assoys.B. Incubation: The incubation mixture contains 0.9 ml phosphate buffer (pH  6.0, O.lfi)  containing 400 pq cosein  plus0.1 ml extract. The casein  in buffer is incubated at  35°C for 5 minutes and  the extract is added. After a further 15 minutesat  35”C,  1 ml of l&i perch&c  acid is added  to stop the reaction. After IO minutes at  room  temperature, the tubes orecentrifuged and  the supernatant  is pipetted off. A blankin  which the perchloric acid  is added before the extract is run fareach extract used.C. Tyrosine estimation: (Udenfriend and Cooper 1952  J .  Biol.  Chem. 196:227):Reogents: (I) I-nitrow-2-naphthol  solution; 0.1% nitrosonaphthol in 95% ethanol. (2) Nitric acid - radium  nitrite;2.6 R nitric acid containing 0.05% sodium nitrite. This moy be conveniently prepared by adding 0.5 ml of 2.5% sodiumnitrite to 24.5 ml of 2.6R  nitric acid immediately before “se.Assay: The reaction mixture contains I ml of supernatcnt  + 0.5 ml nitrosonaphthol wlution  + 0.5 ml nitric acid-sodiumnitrite. Mix and  heat at 55’C  for 30 minutes. Cool, add 5 ml 1,2-dichloroethane  to each tube, mix thoroughly and centrifugeto break the emulsion. The dichloroethane  layer contains the unreocted  nitrosonaphthol. Measure the optical density of theupper layer at 450 qJ. Reagent blanks  and tyrosine standards should be run with each set of assays. - - - Division of Biology,Colifornio  Institute of Technology, Pasadena, California.Eilers,  F.  I.  and G. Fischer. An efficient grinder for In working with Neurospora  ascospaes  we hove found it necessaryoscospores  and mycelium. to develop an efficient method of grinding small samples  of spores.Arnold and Oppenheimer (1950 J. Gen. Fhysiol. 33:432)mounted a gloss syringe bwrel  containing a sample of Chroococcvs  in a lathe, and by forcing the plunger into the slowly turningbarrel  got effective breakage. Using this idea, we have developed an efficient instrument from cornman  and inexpensive materials.In this machine the syringe plunger is mounted in the chuck of CI  high torque stirrer motor through o bolt cemented to a rubberstopper with epoxy resin. The end of the plunger is cemented to the other end of the stopper. This is CI  secure, yet flexible,connection. The tip of the barrel is sealed with epoxy  resin and the barrel is mounted in a chuck mode of heavy-walled rubbertubing (13/16  x 3/B”) which, in t”m,  is fastened by a hose clamp to a wooden block on the end of o threaded rod. This arrange-ment for holding the barrel  olw  is designed to import flexibility in order to prevent excessive breakage of the plunger. The 3/B”threaded rod is aligned with the plunger by D U-shaped brace  of l/4”  steel. A handle at  the end of the rod is “red to turn itthrough the brace, thus advancing the barrel over the plunger. Because of the force necessary to advance the barrel over theplunger, it is necessary to mount the individual parts to CI  sturdy one-inch plywood board.In practice the grinder is loaded by tipping it up  and pipetting the spore suspension  into the barrel. We generally “se100-200 mg of ascospores  in 5 ml of wrspending  medium in CI  IO  ml syringe. The barrel is fitted over the plunger, and oil the airis forced out. The grinder is then laid flat, the motor turned on, and the barrel advanced over the slowly turning plunger. Thehomogenate, which drips slowly from the end of the barrel, can easily be caught in a beaker. A 5 ml sample  in a 10 ml syringegenerally is ground in I5 minutes  if the syringe is close-fitting. In the case of syringes with “nusuolly close tolerances it isnecessary to reduce the closeness of fit by (I few seconds grinding with fine corundum. If the grinder is “red in CI  cold room, andif the plunger is turned slowly, no serious heating occ”rs.By “sing (I 50 ml syringe with a conical tip on the plunger, the grinder works well on mycelium that has  been pre-ground ina blender. - - - Department of Botany, University of Michigan,  Ann Arbor, Michigan.Flavin, M. and C. Slaughter. Methods “red for protein in recent years  we have extracted Neurospora  by hand grindingextraction. with alumina, which releclses  more protein than  any other methodwe  had tried, and we still “se it on a small  stole.  For large scalepreparations we now “se o “laboratory homogenizer” monufclclured  by the Manton-Goulin  Company of Everett, Mass.  This press,which occupies little floor space although it weighs 450 pounds,  extracts 3 times more protein than alumin.  does  and is c1 greatdeal  more  agreeable to “se. A kilogram of freshly harvested mycelium is dispersed as  well as possible  in (I l&ge  blendor into anequal weight of buffer. This chilled suspension can be passed  through the press in 3 minutes. On a larger stole,  the procedurewould probably have to be interrupted, since the extract is coming out clt4VC  after 3 minutes. If the press jams it takes 2 hoursto dismantle and  clean it. W he we  hod no problem with this since we took to stationing a second man  at the bond  wheel, torelease  it the instant the pressure rises at-we 8,ooO  pa”nds/inch2.We hove always ultracentrifuged  Neurospora  extractr  prior to fractionation. We originally started  this because of poor  re-sults with acetone fractionation when centrifugation  was  omitted: a gelatinous precipitate formed a+  low acetone concentrationsond adrorbed  some soluble protein. This procedure seems to be particularly necessary with extracts of stowed  cells. These aregrown with limiting sulfur  ( FI win 1965  Biochem. Biophys. Res. Commun. 20:652);  the cell mass is 35% and the extractableprotein 15% of normal. The extracts ore run for 4 hours in the Spinco  21 rotor. Besides the pellet and the fat pellicle,  it isessential to discard about the top 15% of solution, even though no refractile  difference can be seen. Otherwise, ~loner or laterthe protein will float when a salt fraction is centrifuged, or a viscous solution will be obtained which can’t be passed throughsephadex. - - - Enzyme Section, National Heart  Institute, Bethesda, Maryland.

Assay of proteolytic enzyme(s) from Neurospora crassa

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