PHOSPHORUS METABOLISM IN GROWING CULTURES OFSACCHAROMYCES CEREVISIAE

BERNARD J. KATCHMAN AND WILLIAM 0. FETTYMound Laboratory,' Monsanto Chemical Company, Miamisburg, Ohio

Received for publication November 8, 1954

The inhibitory effects of radiation and chem-icals on the cellular division proceses in yeastand Escherichia coli in the logarithmic phases ofgrowth have been studied in this laboratory. Aninvestigation of phosphorus metabolism wasundertaken as part of a program to correlatemetabolic changes accompanying the inhibitionof cellular division. In order to devise satisfac-tory experimental conditions for determining theeffects of division-inhibitors on the phosphoruscompounds in rapidly-dividing yeast cells, itbecame necessary to know the phosphorus spec-trum of normal growing cultures. However, thepaucity of data on the phosphorus metabolismof rapidly-dividing yeast cells at various stagesof growth made it imperative that this study bemade since it is not unlikely that the phosphoruscontent(s) of a particular fraction(s) might varyduring the logarithmic growth cycle. This im-plies that, although the cells tain a steadyrate of growth and multiplication during the logphase, the concentrations of intermediary me-tabolites of other components of the cell neednot be in steady state.

This paper describes a method for the frac-tionation and pbosphorus analysis of normalyeast in various stages of growth and division.

METIOD

General. Saccharomyces cereuiae was used inall experiments. The cultures were grown asdescribed previously (Spoerl et al., 1954). Ali-quots of the culture were removed for mass andcell-count determinations and phosphorus frac-tionation. Cell counts were made with a hemacy-tometer, and culture mas was determined bymeasurement of dry weights (24 hr at 105 C).

Phosphorus determination. Phosphorus wasdetermined colorimetrically as orthophosphate

1Mound Laboratory is operated by MonsantoChemical Company for the United States AtomicEnergy Commission under Contract Number AT-33-1-GEN-53.

by the method of Fiske and Subbarow (1925) ex-cept that perchloric acid was substituted forsulfuric acid because barium perchlorate ismore soluble than barium sulfate and does notinterfere in the polyphosphate analysis.

In trichloracetic acid extracts, orthophosphatewas estimated colorimetrically by the abovemethod after it was found that there was noessential difference in the results when extractswere analyzed either under the milder conditionsof the Lowry and Lopez (1946) method or byquantitative precipitation of the orthophosphate.

Quantitative precipitation of orthophosphatefrom the trichloracetic acid extract can be ef-fected, when desired, as the magnesium am-monium phosphate (Kolthoff and Sandell,1945) or by the method of Delory (1938). Theseprecipitates dissolve readily in dilute acid, andthey can be estimated colorimetrically.

Total labile phosphorus must be heated in 1N acid for 10-15 min at 100 C to effect quantita-tive hydrolysis. The orthophosphate from suchhydrolyzed extracts was determined colorimet-rically.

Total phosphorus was estimated colorimet-rically after conversion of the sample to ortho-phosphate by digestion with perchloric acid for1 hour at 130-160 C (LePage, 1949).Polyphosphate can be precipitated from solu-

tion by barium ion at pH 2.5 and at pH 4.5. Theprecipitates dissolve readily in 1 N HCl and areconverted to orthophosphate by treating as forlabile phosphorus or total phosphorus. Theprecipitates are 100 per cent labile phosphorus,and they are not contaminated with orthophos-phate or stable organic phosphorus compounds.

Pentosenucleic acid was estimated from thepentose content of extracts by the orcinol methodof Ogur and Rosen (1950), the phosphorus con-tent, and the ultraviolet absorption at 260 m,u.As a reference standard, purified commercialpreparations of yeast nucleic acid (SchwarzLaboratories, N. Y.; Nutritional Biochemicals,

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Cleveland) were used which assayed 2 moles ofribose for four moles of phosphorus with an ex-tinction per mole of phosphorus of 9,600.

Desoxypentosenucleic acid was estimated fromthe total phosphorus and the ultraviolet absorp-tion at 260 m,u. Neither the Stumpf (1947) northe Dische (1930) colorimetric assays for desoxy-pentosenucleic acid on the yeast extracts weresufficiently stable to permit quantitative deter-mination by these methods.

Extration and partin of pho#phoru fractios.The methods used for extraction and partitionof phosphorus fractions from yeast cultures area modification of procedures reported in theliterature (Wiame, 1949; Juni et al., 1948;Schmidt and Tnhauser, 1945) which wererevised when found unstisfactory for the pur-poses of the present work.The effectiveness of cold (4 C) trichloracetic

acid as an extracting agent was investigatedover a concentration range of 5-40 per cent forthe first one hour extraction, followed by twoadditional one hour extractions with cold 10 percent trichloracetic acid. Calculated amounts of60 per cent trichloracetic acid were added toyeast suspensions to give the final concentrationdesired. Since 90 per cent of the total acidsoluble phosphorus is removed during the firsthour of extraction, the effect of trichloraceticacid during this period was investigated. Resultsshowed that the amounts of orthophosphate andlipid phosphorus extracted were unaffected overthe entire range of acid concentration. However,5 per cent trichloracetic acid does not extractall of the acid soluble polyphosphates (Wiame,1949), and any unextracted polyphosphate of thisfraction is found later with the acid insolublepolyphosphate fraction. Ten per cent trichlorace-tic acid provides maimum extraction of acidsoluble polyphosphates with no increase or de-crease over the range 10-40 per cent. The acidinsoluble polyphosphate fraction extracted alsoremains constant over this range. The most pro-nounced effect of acid concentration appears tobe on the nucleic acid fraction as indicated by agradual increase in the 260 m,u absorption of thetrichloracetic acid extract and the acid solubleorganic phosphorus, and a decrease in the nucleicacid fraction. However, the greatest changesoccur at 30 per cent or higher acid concentration.

In experiments other than those mentionedabove, the efficiency of extraction was found tobe a function not only of the concentration ofacid but of the total amount of acid for a par-ticular sample weight. Routinely, 200-300 mg(dry weight) samples of yeast were made 10 percent with respect to trichloracetic acid, and600-800 mg samples were made 15 per cent withrespect to trichloracetic acid for the first hour ofextraction. The two subsequent extractions ofone hour duration were carried out in 10 per centtrichloracetic acid. All extractions and centrifuga-tions were carried out at 4 C.The trichloracetic acid residue was extracted

twice with 95 per cent ethyl alcohol at 27 C andthree times with 3:1 alcohol-ether mixture at80 C for 3 min to obtain the lipid-phosphorusfraction. The extracted residue was dried to afine powder. This residue contains the insolublepolyphosphate and the nucleic acids. The use of1 N NaOH at 37 C for extended periods of timeto solubilize the pentosenucleic acids (Schmidtand Thannhauser, 1945; Juni et al., 1948) wasincongruous with the known lability of poly-phosphate. At pH 8 and 27 C, 90 per cent of thepentosenucleic acid is immeiately solubilized;consequently, such harsh treatment as 1 N alkalifor 24 hours at 37 C is unnecessary. The driedresidue was suspended in 1 N NaOH at 27 C, im-mediately centrifuged, and washed twice with 1N NaOH. This quantitatively solubilizes thepentosenucleic acid and the polyphosphate.Since no orthophosphate could be detected, theseconditions were satisfactory for solubilizing pen-tosenucleic acid without hydrolysis of the poly-phosphate. The alkaline extract was adjusted topH 4.5; the slight precipitate which forms con-tains no phosphorus and is centrifuged. The super-natant liquid contains degraded pentosenucleicacid and the insoluble polyphosphate. The poly-phosphate is precipitated with barium ion and isfree of 260 m,u absorbing material and carboncontamination. The desoxyribosenucleic acidwhich remained in the alkaline residue was es-timated after hydrolyses for 5 min in 1 N HClat 100 C.The method is illustrated by means of a typical

experiment (figure 1). An aliquot of yeast cul-

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WASHE CELLSEXTRACTION 3 TIMES TRICHLORO-ACETIC ACID; CENTRIFUGATION

SUPER ATANT

ORTHO |TOTAL | BJ!PPT7MIN. 86"*PPT (PH 2.S)

(PH 4.5)POLYPHOSPHATE

FRACTIONS

SUPERNATANTLIPID-P

RESIDUE

ALCOHOL- ETHEREXTRACTION;CENTRIFUGAT ION

RESIDUE

NGOH

ALKALINE SUPERNATANT ALKALINE RESIDUE

ADJUST TO PH 4.5; IN. HCICENTRIFUGATION g MIJN.

CENTRIFUGATION

SUPERNATANT RESIDUE SUPERNATANT RESIDUE(DISCARD) 'DESOXYRIBOSE- (DISCARD)

NUCLEIC ACID'

PENTOSENUCLEIC Si++ PPT., PH 4.5ACV (POLYPHOSPHATE

WHEN PRESEN1t)

Figure 1. Analytical fractionation scheme

ture, approximately 700 mg dry weight, iscentrifuged, washed twice with cold distilledwater, suspended in water at 27 C for 10 min,and centrifuged at 4 C. To these cells are added2.5 ml of 60 per cent cold trichloracetic acid andsufficient cold water, with stirring, to make afinal volume of 10 ml. After 1 hour at 4 C thesuspension is centrifuged for 10 min and the cellsreextracted twice (each for one hour) with S mlportions of cold 10 per cent trichloracetic acidat 4 C. The three extracts are collected and madeto 20 ml with cold water. This is the acid solublefraction. The residue is suspended in 95 per centethyl alcohol at 27 C for 30 min and centrifuedat 27 C. The alcohol extrction is repeated. Theresidue is extracted 3 times with 5 ml of 3:1alcohol-ether mixture on a water bath at 80 Cfor 3 min and centrifuged at 27 C. The combinedsupernatants, approximately 25-30 ml, are desig-nated as the lipid-phosphorus fraction. Theresidue from lipid extraction is warmed on a waterbath and agitated with a stirring rod to removeresidual organic solvent and to provide a finepowder. This residue is blended in 10 ml of 1 NNaOH at 27 C with a stiring rod to give a

homogeneous suspension which is centrifuged.The alkaline insoluble residue is washed twicewith 1 ml portions of 1 N NaOH, and the com-bined supernatant and washing are collected.The alkaline extract is treated with concentratedHCI by dropwise addition until a slight turbiditypersists (0.7 ml), and then 1 ml of 3 M acetatebuffer pH 4.5 is added. The turbid suspension iscentrifuged (18,000 xG) for 10 min, and theresidue is washed twice with 1 ml portions ofacetate buffer. Supernatant and washing are col-lected, made to volume (20-25 ml), and desig-nated as the pentosenucleic acid fraction. Thealkali insoluble residue is suspended in 5 ml of 1N HCI and hydrolyzed for 5 min at 100 C. Thefinal acid insoluble residue contains no phos-phorus.

Acid soluble extract. The cold extract is analyzedimmediately for orthophosphate, total labilephosphate, and total phosphorus. Aliquots areremoved for precipitation.

Orthophosphate: Three milliliters of acid ex-tract (500 ug P) are treated with 1 ml magnesiamixture (Kolthoff and Sandell, 1945) at 4 C,tben concentrated NH4OH is slowly added drop-

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wise until the solution is alkaline to phenol-phthalein, after which a large excess of NH4OH(2-3 ml) is added. The sample is stored over-night at 4 C, then centrifuged, and the precipi-tate is washed twice with cold dilute NH4OH(1:10). The precipitate is dissolved with 10 ml1 N HCl and analyzed. These precipitates showsome contamination of labile phosphorus. How-ever, if polyphosphate is first removed by pre-cipitation with barium ion (Jones, 1942), thenorthophosphate may be precipitated quantita-tively and free of labile phosphorus.

Polyphosphates: Two milliliters of acid ex-tract are neutralized in the cold with 5 N NaOH,made slightly acid to methyl orange (1 drop 1N HCI), and then 1 ml of 3 M acetate buffer pH4.5 and barium ion are added with stirring toprecipitate polyphosphate. A second aliquot istreated similly but is precipitated with bariumat pH 2.5. The solutions are stored overnight at4 C, centrifuged, and the precipitates are washedtwice with dilute BaCl2 solution (1 g/liter). Theprecipitates are dissolved in 5 ml 1 N HCI andanalyzed. These precipitates are 100 per centinorganic labile phosphorus.

Pyrophosphate: Attempts to separate quanti-tatively pyrophosphate from these extracts havegiven such ambiguous results that all of the labilephosphorus other than polyphosphate is desig-nated as acid soluble labile phosphorus. Attemptsto precipitate pyrophosphate with manganousion by the Jones method (1942), with cadmiumion by the method of Cohn and Kolthoff (1942),

TABLE 1Steady-state phosphorus fractions

Num-Concenmtionber of

Fraction Concentraton RunsmigP/100 mg' Aver-aged

Acid soluble pH 2.5 bariumprecipitatet.............. 0.472 i 0.014 26

Acid soluble residual la-bilet.................... 0.230 4 0.021 26

Acid soluble stable organic§ 0.125 4 0.010 51Lipid. 0.137 i 0.002 43

* 95% confidence interval for the true mean.t Inorganic polyphosphate average chain length

about 20.t Residual labile-(total-P 10 min)-(ortho +

pH 2.5 barium precipitate).§ Stableorganic-(totalacid-soluble-P)-(total-

P 10 min).

and with zinc ion by the Bell method (1947)proved unsuccessful.

Lipid-phosphorus fraction: Two milliliter ali-quots (60 jg P) of a 30 ml alcohol-ether extractare pipetted into 25 ml volumetric flasks, a fewdrops of water are added, and the organic solventsare evaporated on a boiling water bath. Onemilliliter of 70 per cent perchloric acid is added,and the sample is digested at 130-160 C as forthe total phosphorus determination.

Pentosenucleic acid fraction: The total phos-phorus (1:5 dilution) and the ultraviolet ab-sorption at 260 m,u (1:100 dilution) of the sampleare obtained by direct analysis of diluted aliquotsof the extracts. The polyphosphate content isobtained by barium ion precipitation of an ali-quot of the extract. The difference between thetotal phosphorus of the extract and the polyphos-phate content gives the pentosenucleic acidphosphorus content. This is valid because theextinction per mole of phosphorus is close to thetheoretical for pentosenucleic acid, and the samplepentosenucleic acid calculated from ribose analy-ses and phosphorus gives excellent agreement.The Delory method (1938) and the magnesiamethod failed to give a test for orthophosphatein these extracts.

RESULTS

The growth characteristics of S. ceraisia,under the conditions of the experiments reportedhere, are shown in figure 3. These cultures main-tain logarithmic rates of growth (increase inmas) and multiplication to a culture count ofabout 6.8 X 107 cells/ml, at which point thegeneration time increases rapidly as the cells goout of the log phase, until the cultures reach astationary phase at about 11.2-12.2 X 107cells/ml. The data reported below cover the rangeof cell concentrations 4.5 X 10 to 13.5 X 107cells/ml. Throughout this range of concentrations,which covers part of the logarithmic growth phaseand the phase of negative acceleration up to thestationary phase, the individual cell mass re-mains constant at a value of 37 IAsg. This makesit possible to report the data as mg P/100 mgdry weight rather than on a per cell basis. Thelower limit of cell concentration of 4.5 X 106cells/ml was chosen because it becomes ex-ceedingly cumbersome below this concentrationto obtain a sufficient number of cells for accurateanalyses.Table 1 summarizes the results obtained for

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those fractions which occur in a steady-state cellconcentration. This is indicated by a linear func-tion with zero slope within the limits of the ex-perimental error over the entire range of growthstudied. With the exception of orthophosphate,all acid soluble phosphorus compounds are found

1.2 ~\

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to be in a steady-state concentration. The pH2.5 barium precipitate is made up of a linear in-organic polyphosphate of an average chain lengthof about 20 phosphate units as determined bytitration, which is not at variance with the re-sults of Ebel (1952) and Talley and Katchman

NO. CELLS/ML. (x 107)Figure 2. Variation in cellular concentration of pentosenucleic acid and orthophosphate as a function

of growth.The dotted lines represent the 95 per cent confidence interval for the individual experiments. The

least squares regression line was determined for pentosenucleic acid phosphorus from 32 experimentsand the orthophosphate line from 51 experiments.

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BERNARD J. KATCHMAN AND WILLIAM 0. FETTY

(unpublished data). The residual labile phosphorusfraction would be a measure of such compoundsas tripoly- and trimetaphosphate (Ebel, 1952;Kornberg and Kornberg, 1954), tetrapolyphos-phate (Ebel, 1952), as well as certain organic

phosphorus compounds such as adenosine tri-phosphate, etc. Pyrophosphate could not be pre-cipitated from the acid extracts and probably isnot present as such in aerobic yeast cultures(Ebel, 1952). Since the pH 4.5 barium precipitate

CELL CT DRY WT.(X107j)( mg/ml)30-

.-0 LOG PHASE

5D0

4.0_~~~~~~~~

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-I.5 GROWT CURV

-LO-.9-.8-.7-.6-.5

POLYPHOSPHATE-.4

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I~~~~~~~~~~~~-.10-.09

-.07

NEGATIVEACC. -STATIONARY -

IIQ7x10

-4.5

L!AIDJNSOL.FRACIO2N

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.2

.15

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It . _ l T _ 'I I

1 2 3 4 5 6I . II I

7 8 9TIME (HRS)

I I II10 II 12

Figure S. Relationship between growth curve and occurrence of the insoluble polyphosphate frac-tion.The curve for cell counts vs mg P/100 mg dry weight was transformed to a mg P vs time curve by

use of the growth curve. The left hand ordinate scales representing cell count and mg dry weight vstime are logarithmic scales. The right hand ordinate scale is linear.

8-

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PHOSPHORUS METABOLISM IN S. CEREVISIAE

should contain the total polyphosphates (includ-ing the polyphosphates of pH 2.5 fraction), at-tempts were made to estimate the amount of thesmaller chains by subtracting the value of thepH 2.5 fraction from the pH 4.5 fraction. How-ever, the precipitation of pH 4.5 fraction was tooerratic to permit such an analysis. The stableorganic fraction is a measure of the organic phos-phorus compounds which contain phosphorusbound to carbon that is not hydrolyzed in 10 minat 100 C in 1 N acid, as for exple adenosinemonophosphate, etc. The results obtained forthe desoxyribosenucleic acid fraction were notincluded in table 1 because of the exceedinglysmall amount found and because the difficultiesencountered in making accurate measurementsdid not provide data amenable to statisticalanalysis. However, the desoxyribosenucleic acidcontent could be approximated at 0.050 mgP/100 mg as a steady-state concentration. Lipid-phosphorus, in the amount 0.137 mg P/100 mgwas found in a steady-state concentration.The phosphorus concentration of a fration

in the steady-state means that the amount ofthat fraction per cell is constant. The concentra-tion of these intracellular phosphorus compounds,both inorganic and organic, appears to be in-dependent of the physiological state of -aerobiccultures, and does not reflect changes in metabolicactivity-such as decreased rate of growth andmultiplication. The role which these substances(acid soluble polyphosphates, labile-, stable-,desoxyribosenucleic acid-, and lipid-phosphorus)play in taining growth rate is not apparent,and no reason is evident for maitaining theeparticular steady-state concentrations fromgeneration to generation. However, there is anabundance of information in the literature whichhas established the importance of the phos-phorylated organic metabolites and desoxy-ribosenucleic acid in the metabolism of yeastcels. A relationship would be expected in whichthe rate of synthesis parallels the rate of growth.Why the acid soluble inorganio polyphosphatesand the lipid-phosphorus fractions fall into thispattern is not evident.

Figure 2 shows that the orthophosphate andthe pentosenucleic acid fractions per cell con-tinuously decrease in amount as the culture ages.The rate of decrease is constant, and, as for thesteady-state fractions, neither the rate of de-crease nor the decreased amount per cell seems to

be affected by or related to the change in meta-bolic activity. However, the fact that there is adecreased amount (per cell) of the orthophosphateand the pentosenucleic acid as the culture agesmust mean that these substances are activelyinvolved in growth and division processes butthat the amount available to the cell, either fromprior synthesis of pentosenucleic acid or from themedium (orthophosphate), is over and abovethat required to sustain logarithmic rates ofgrowth and division. The fact that in growingcultures the amount of pentosenucleic acid percell is not constant is perhaps the reason thatconflicting data are found in the literature as tothe actual amount of pentosenucleic acid per cellsince this value would vary depending upon thephysiological time at which the measurementwas made.A most striling change occurs in the insoluble

inorganic polyphosphate fraction during cultureaging. In figure 3, it can be seen that below 1 X107 cells/ml there is no insoluble inorganic poly-phosphate present in the cell, within the limitsof the precipitation method used. However, be-tween 1 X 107 and 7 X 107 cells/ml there is arapid build-up of this polyphosphate fraction toabout 0.4 mg P/100 mg, and from this point tobetween 5-8 X 107 cells/ml the amount levelsoff and then decreases. The data suggest that ametabolic activity of the cell (synthesis of in-soluble polyphosphate) undergoes change coin-cidental with an increase in generation time. It isnot clear, as yet, either what induces polyphos-phate synthesis or just how it may be related tothe change in generation time. Nor does the rateof decrease of orthophosphate nor the rate of de-crease of pentosenucleic acid in any way indicateany relationship to the polyphosphate build-up.The source of the orthophosphate monomer pre-cursor of the insoluble polyphosphate is notevident from these studies.

DISCUSSION

For comparative biochemical studies wherenormal and treated cultures are to be compared,it is not enough to show that a change has oc-curred in a particular fraction or component bycomparng controls and experimental cultures; itmust be shown that these changes occur either atcomparable physiological times or in fractionswhich are in steady-state concentrations through-out the growth period. It is obvious from the

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data reported herein that unless orthophosphate,pentosenucleic acid, and insoluble polyphosphateare compared at the same physiological time, anerroneous change would be recorded whichwould in no way be related to the effects pur-ported to be under investigation.The nature of the polyphosphates and the role

which they play in yeast metabolism have beenobscure for many years. Recently, it has beenshown (Juni et al., 1947; Wiame, 1949) that thereare a soluble and an insoluble polyphosphatefraction in yeast. Phosphorus turnover studies(Juni et al., 1947) and metabolic studies (Wliame,1949) have shown that these two fractions havedifferent metabolic activities, the insoluble frac-tion being more active. This is substantiated bythe results reported here. It is shown that thesoluble inorganic polyphosphate fractions main-tain a steady-state concentration from genera-tion to generation while the insoluble polyphos-phate fraction appears only in the later stages oflogarithmic growth in a nonsteady-state concen-tration independent of the concentration of thesoluble polyphosphates. Thus, there can be nodoubt that these two polyphosphate fractionsare different and have differing metabolic activi-ties.The fact that the insoluble polyphosphate,

which is not present over a portion of the growthphase, builds up, levels off, and then decreases inconcentration as the generation time increasesmight indicate its role as a growth-regulator, con-trolling the generation time in the cell. Wiame(1949) has reported that the build-up or break-down of the insoluble polyphosphate is reflectedin the intracellular orthophosphate concentra-tion. Such a relationship between orthophosphateand insoluble polyphosphate is not apparentfrom these results. This could mean that morethan one pathway exists for the synthesis andbreakdown of the insoluble inorganic polyphos-phate.

Synthetic inorganic polyphosphates are knownto complex an unusually wide range of inorganiccations (Van Wazer and Campanella, 1950) suchas calcium, magnesium, sodium, etc. as well asproteins (Briggs, 1940; Gustavson and Larsson,1951; Katchman and Van Wazer, 1954) when inthe cationic form. In a recent report (Katchmanand Van Wazer, 1954), it was postulated that theartificial, though functional, designation of thesoluble and insoluble polyphosphate fractions in

yeast which arises from trichloracetic acid ex-traction could be due to the different mode ofprotein complexing dependent upon polyphos-phate chain length. These experiments suggestthat in addition to the trimeta-, tripoly-, andtetrapolyphosphates and the polyphosphateknown to be present in the acid soluble fraction ofyeast, the insoluble polyphosphate might be apolyphosphate compound with an average chainlength substantially greater than 20. Physico-chemical investigations on the insoluble poly-phosphate fraction are being carried out in orderto establish the identity of this material. It seemsreasonable to assume that the differing metabolicactivities of these polyphosphate fractions maybe due to the differences in complexing proper-ties which arise from differences in chain length.It has been suggested by Lindegren (1949) thatthe polyphosphates in yeast might control growthby their ability to complex or deactivate proteinsor enzymes necessary in intermediary metabo-lism. In addition, it must be considered that thepolyphosphates form complexes with monovalentas well as polyvalent cations, many of which areknown to be required to maintain metabolicactivity (Lehninger, 1950) and therefore mightalso function as growth and division regulatorsby controlling the availability of esential cationsas well as enzymes.

ACKNOWLEDGMEN

We wish to acknowledge the asistance of Mr.Kenneth A. Busch in performing the statisticalanalyses and in constructing the graphs.

SUMMARY

A method for fractionation and phosphorusanalysis of yeast cultures in various stages ofgrowth and division is described.The results of the phosphorus analyses fall

into three distinct patters: (a) steady-stateconcentration fractions, in which the amountper cell is constant along the growth curve, in-dlude the inorganic polyphosphate, the residuallabile and stable organic phosphorus of the tri-chloracetic acid extract and the lipid and thedesoxyribosenucleic acid phosphorus; (b) frac-tions, which decrease continuously with a con-stant rate, include ortho and pentosenucleic acidphosphorus; and (c) the acid insoluble polyphos-phate fraction which builds up in the later stagesof logarithmic growth.

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It has been emphasized that erroneous con-clusions might be drawn from simple compansonsbetween control and experimental cultures un-less due consideration is given to the physiolog-ical state of the cultures.The possible role of the insoluble polyphos-

phates of yeast cultures is discussed.

REFERENCES

BELL, R. N. 1947 Estimation of triphosphoricand pyrophosphoric acids in the presence ofortho- and metaphosphoric acids. Ind. Eng.Chem., Anal. Ed., 19, 97-100.

BRIGGS, D. R. 1940 The metaphosphoric acid-protein reaction. J. Biol. Chem., 132, 261-272.

COHN, G., AND KOLTHOFF, I. M. 1942 Deter-mination of pyrophosphate. Ind. Eng.Chem., Anal. Ed., 14, 886-890.

DELORY, G. E. 1938 Determination of phos-phate in the presence of interfering sub-stances. Biochem. J. (London), 32, 1161-1162.

DISCHE, Z. 1930 tJber einige neue charakteris-tische farbreaktionen der thymonukleinsaure.Mikrochemie, 8, 4-32.

EBEL, J. P. 1952 Etude chromatographique etpotentiometrique des polyphosphates delevure. Bull. soc. chim. biol., 34, 330-335.

FISKE, C. H., AND SUBBAROW, Y. 1925 Thecolorimetric determination of phosphorus.J. Biol. Chem., 66, 375-400.

GUSTAVSON, K. H., AND LARs8oN, A. 1951 Theinteraction of polymetaphosphates with hideprotein. Acta Chem. Scand., 5, 1221-1243.

JONES, L. T. 1942 Estimation of ortho-, pyro-,meta-, and polyphosphates in the presenceof one another. Ind. Eng. Chem., Anal. Ed.,14, 536-542.

JUNI, E., KAMEN, M. D., SPIEGELMAN, S., ANDWIAME, J. 1947 Physiological heterogeneityof metaphosphate in yeast. Nature, 160,717-718.

JUNI, E., KAMEN, M. D., REINER, J. M., ANDSPIEGETMAN, S. 1948 Turnover and dis-tribution of phosphate compounds in yeastmetabolism. Arch. Biochem., 18, 387.

KATCHMAN, B. J., AND VAN WAZER, J. R. 1954The soluble and insoluble polyphosphates inyeast. Biochim. et Biophys. Acta, 14, 445-446.

KOLTHOFF, I. M., AND SANDELL, E. B. 1945Determination of phosphorus, pp. 390-397.In Textbook of quantitative inorganic analysis.The Macmillan Co., New York.

KORNBERG, S. R., AND KORNBERG, A. 1954Inorganic tripolyphosphate and trimetaphos-phate in yeast extracts. Federation Proc.,13, 244.

LEHNINGER, A. L. 1950 Role of metal ions inenzyme systems. Physiol. Revs., 30, 393-429.

LEPAGE, G. A. 1949 Methods for the analysisof phosphorylated intermediates, pp. 190.In Manometric techniques and related methodsfor the study of tissue metabolism. Edited byW. W. Umbreit, R. H. Burris, and J. F. Stauf-fer. Burgess Publishing Co., Minneapolis,Minn.

LINDEGREN, C. C. 1949 The yeast cell, pp. 29-40.Educational Publishers, Inc., St. Louis, Mo.

LOWRY, 0. H., AND LOPEZ, J. A. 1946 The de-termination of inorganic phosphate in thepresence of labile phosphate esters. J.Biol. Chem., 162, 421-428.

OGUR, M., AND RoSEN, G. 1950 The extractionand estimation of desoxypentosenucleic acidand pentosenucleic acid. Arch. Biochem.,25, 262-276.

SCHMIDT, G., AND THANNHAUSER, S. J. 1945 Amethod for the determination of desoxyribose-nucleic acid, ribonucleic acid and phosphor-proteins in animal tissues. J. Biol. Chem.,161, 83-89.

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