Procedure for preparing permeabilized hyphae for enzyme analysis

Gujar, V. P.

Schmit, J. C.

Fungal Genetics Reports

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V. P. Gujar

J. C. Schmit

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Procedure for preparing permeabilized hyphae for enzyme assays

Kansas State University

Fungal Genetics Reports Volume 30 Article 12 Procedure for preparing permeabilized hyphae for enzyme assays V. P. Gujar J. C. Schmit 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 Gujar, V. P., and J.C. Schmit (1983) "Procedure for preparing permeabilized hyphae for enzyme assays," Fungal Genetics Reports: Vol. 30, Article 12. https://doi.org/10.4148/1941-4765.1627 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. Procedure for preparing permeabilized hyphae for enzyme assays Abstract Procedure for preparing permeabilized hyphae for enzyme analysis. This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol30/iss1/12 Flint, H.J., S. Dible  and H. KacserA method for achieving prolonged nutrientlimited growth of Neurospora mycelium.It is difficult to achieve sustained growth undersupplement limited conditions of filamentous fungi suchas Neurospora which cannot easily be grown in a chemo-stat. Reducing the initial concentration of a nutrientprovided to batch cultures usually results in a rapidlydeclining supplement concentration and succeeds only inadvancing the time at which growth ceases. This seldomproduces experimentally amenable variation in growthrate. In order to study the effects of prolonged growthamino acid we have adapted the  of amino acid auxotrophic mutants at low rates of provision of the required   . .  approach of continuously supplying fresh medium, supplemented with the desired amino acid concentration tobatch cultures. The method described should prove applicable to other nutrient limitation conditions such asnitrogen or carbon starvation. 0.1 -/v channel proportioning pump. Pump tubing was presteril-ized by passing a 2% solution of hypochlorite, followedby sterile distilled water. The flasks were shaken at200 rpm on a New Brunswick gyrorotary shaker in 29°Cconstant temperature room for periods not exceeding.o.,-'./0 45 h, or a final culture volume of around 600 ml. In7the experiments shown in Fig. 1 conidia from an arg-5strain were inoculated into replicate flasks containingVogel's medium supplemented with 2.25% (w/v) glucoseand 0.12 mM arginine, and supplied at a constant rate/~? 0.2  - d ./i.I0with fresh Vogel's/glucose medium containing variousconcentrations of arginine. Once the initial supplyf ,/LA-----------'of arginine had been exhausted, growth occurred at alinear rate proportional to the concentration ofarginine in the feeding solution. Mycelium growing0.I - J.&#+  ..-• 7.at a linear rate cannot represent a true metabolicsteady state, as is established under conditions ofexponential growth. Nevertheless such experiments canyield valuable information on changes in enzyme syn-Replicate 1 L round, flat bottomed flasks were used, having three equally spaced 2 cm identations toimprove agitation and aeration. These contained an initial volume of 200 ml of medium, inoculated witharound 1O7 conidia. The flasks were fitted with silicone rubber stoppers carrying a foil capped vent and aglass tube in contact with the medium through which acombined stream of sterile air (0.42 ml/min) and freshmedium (0.16 ml/min) was pumped by means of a multi-Fiqure 1. -- Growth of an arg-5 inoculatedthesis (in this case the derepression of amino acidsynthetic enzymes - Flint et al., in preparation) andin macromolecular svnthesis during prolonqed periods ofnutrient limited growth. A somewhat different 'fedbatch' culture technique, suitable for growth andsampling of germinating conidia, has been described by 1234-1241) forinto-200 ml of medium containing 0.12 mM Limon-Lason et al ((1977) BBRC, 78:arginine and supplied continuously with medium the study of nitrogen and carbon limited conditionssupplemented with arginine at the followingconcentrations: 0.20 mM (∇) 0.12 mM (Ο) 0.04 Using the conditions described above the growth of(0) 0.0 mM (0).  Cultures that received no a wild strain in minimal medium was shown to be exponen-supply of fresh medium, but were initially tial and the rate independent of culture volume oversupplemented with 0.12 mM (9)  or 2.0 mM (I) the maximum duration of each experiment. Thus the com-arginine are shown for comparison. Full de- bination of vigorous agitation and forced air ensurestails are described in the text. adequate aeration throughout, despite the change inculture volume.Department of Genetics, University of Edinburgh, Edinburgh EH9 3JN, U.K.Gujar,  V.P. and J.C. SchmitProcedure for preparing permeabilizedhyphae for enzyme assays.Permeabilization procedures have been developed formeasuring many enzyme activities in dormant and germin-nating  conidia (Basabe et at., 1979, Anal. Biochem. 92:356; Christensen and Schmit, 1980,  J. Bacteriol., 144:983). We have modified this procedure in order tomeasure enzyme activities in permeabilized hyphae. Theprotocol used to prepare permeabilized hyphae for glutamicacid decarboxylase (GAD) assays is given below.Vegetative hyphae and aerial hyphae were obtained from standing cultures (Table I). The vegetative hyphaeformed a pad on the surface of the liquid medium and, after a few days, aerial hyphae grew from the mycelialpad. The aerial hyphae and then the mycelial pad were removed from the test tube with a sterile spatula. Imme-diately the material was agitated on a vortex mixer for 2 min. with 5.5 ml of the permeabilization solution.The permeabilization solution contained 0.5 ml of toluene:methanol (1:4 v/v) and 5 ml of 20 mM potassium phos-TABLE I.GADa activities in conidiating culturesbDays‘ Specific Activitynmoles CO2 min-1  (mg lyophilized weight)-'Vegetative hyphae            Aerial hyphae2 0.323 t O.O05d4 0.885 * 0.007 0.735 i 0.02aGAD was assayed by measuring the amount of 14CO2 released fromDL[1-14C] glutamic acid (Christensen and Schmit, 1980 J. Bacteriol.144: 983).bStanding conidiating cultures of nada (FGSC #2688) were grown intest tubes (18 x 150) on 5 ml of liquid complete medium containing1% casamino acids, 0.5% yeast extract, 2% sucrose, 1% glycerol andVogel salts (Vogel 1964, Am. Nat. 98: 435) at 25°C.cDays after inoculation with 5 x l05 conidia/tube.dThe assays were done in duplicate.phate (pH 6.8), 10 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride (PMSF) and 1 mMbenzimidine hydrochloride (BHCl). Thepermeabilized cells were collected bycentrifugation, transferred to preweigheddisposable micro test tubes (1.5 ml) andfrozen with liquid nitrogen. The frozencells were lyophilized in a Speed-vacConcentrator (SAVANT). The lyophilizedcells were weighed and ground to a finepowder. Small samples, 5-15 mg of cells,were ground on a cold spot plate with aglass rod whereas larger samples wereground with a cold mortar and pestle.The ground cells were suspended in abuffer (20 mM potassium phosphate (pH6.8), 10 mM EDTA, 1 mM PMSF, 1 mM BHCl)to give a final concentration of 20 mg/mlof lyophilized mycelia.Aliquots of the crude cell suspen-sion were transferred directly to theGAD assay mixture with an automaticpipetter. The pipet tips were cut offto ensure that a uniform sample wastransferred. The specific activitiesof GAD in vegetative hyphae and aerialhyphae were determined (Table I). Eachassay was done in duplicate and the de-viation between duplicates was less than3%. This experiment has been repeatedseveral times and the specific activi-ties of duplicate assay;  always variedless than 10%We have found that this procedure for measuring enzyme activities in hyphae has the following advantages,as compared to preparing cell free extracts.1. The permeabilization of the hyphae assures that all the enzyme activity, intraorganellar as well ascytosolic, is accessible for measurement.2. Small molecules are removed rapidly during permeabilization; thus, dialysis is not necessary.3. The cell interior can be rapidly  exposed to EDTA and protease inhibitors minimizing enzyme degradation4. Lyophilized cells can be weighed to caclulate specific activities.- Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901.Pall, M.L.Rapid sampling and collection ofNeurospora mycelial suspensions.Many experiments with Neurospora involve the taking ofmultiple samples of mycelial suspensions as a function of timeafter various treatments. One of the difficulties in workingwith mycelia is that they are often too coarse to be pipettedeasily. Furthermore, because parameters to be measured maychange rapidly, it may be important to collect the samples veryrapidly to avoid changes during collection.This note describes procedures we have found useful bath for rapid sampling of small aliquots of Neuro-spora mycelial suspensions and for rapid filtration of such samples.Neurospora liquid shaking cultures (l-24 h, 25" in liquid minimal medium, density of about 1 mg dry wt/ml)can be sampled using a variety of dispensers which fit into 24/40 ground joint erlenmeyer flasks (variouslynamed "Repeater Pipets", "Tipets", "Prepipettors', "Tilt-a-Pets", etc.). One can typically take 10 ml samplesvarying by 15% with such medium density culture as described above.Such samples can be collected on Whatman GF/A glass fiber filters. We typically collect such samples on2.4 cm diameter filters using Millipore or similar filter holders having sintered glass bases. The samplescan then be placed into TCA, PCA, ethanol or liquid nitrogen for extraction or rapid freezing. If it is notnecessary to wash the samples, it is not difficult to take a sample, filter it and move it into TCA-containingtube in 3 to 5 seconds. We have shown that such samples may have ATP/ADP ratios of 7, showing that they retaina high energy charge throughout the collection procedure.The GF/A filters are very fast in filtering mycelia of Neurospora and other fungi. Conidia, however, tendto clog the filters fairly rapidly. Consequently, it is important that conidia used to grow the mycelia arerelatively fresh with good germination frequencies to avoid slowed filtration due to ungerminated conidia.(Supported by NIH grant GM-24368) Programs in Genetics and Cell Biologyand Biochemisty/Biophysics.) MolecularGenetics, Washington State University, Pullman, Washington 99164-4350.1 6

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Procedure for preparing permeabilized hyphae for enzyme assays

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