Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum

J. Mol. Biol. (1977)

116, 161-169

Ammonia Determines the Choice of Morphogenetic Pathways in Dictyostelium discoideum JOELSCHINDLERAND MAURICE SUSSMAN Department of Life Sciences University of Pittsburgh Pittsburgh, Pa. 15260, U.S.A. (Received 10 May 1977, and in revised form 12 July

1977)

Previous work (Newell et al., 1969) had indicated that the transformation of a newly formed multicellular aggregate into a migrating slug rather than a fruiting body depended on the local accumulation of a diffusible metabolite excreted before and during aggregation. The subsequent shift back to the fruiting mode could be accounted for by the disappearance or removal of the metabolite. All these morphogenetic transformations were associated with profound and consistent changes in the patterns of enzyme accumulation and disappearance (Sussman et al., 1975). In the present study, evidence is presented to support the conclusion that the metabolite in question is NH,. Thus : (a) Cell populations accumulated and excreted significant NH: before and during cell aggregation under the conditions

amounts of NH, employed.

$

(b) Excretory products collected from such cells induced newly formed aggregates to develop into migrating slugs under conditions which otherwise would permit them to construct fruiting bodies directly. The inducing activity of such preparations was precisely correlated with the NH, + NH: concentration and was destroyed by incorporation of the NH, into glutamic acid in the presence of glutamate dehydrogenase. (c) The slug-inducing activities of the extracellular metabolite preparations and purified fractions thereof were mimicked by equimolar solutions of ammonium carbonate at, identical pH values. (d) Isolated migrating slugs treated with 0.5~1 samples of a reaction mixture containing glutamate dehydrogenase, a-ketoglutarate and NADH immediately stopped migrating and constructed fruiting bodies. Samples lacking a single component or containing boiled enzyme were ineffective. (e) By several criteria, the biological and biochemical performances of autoinduced slugs and of those induced by the metabolite preparations or by ammonium carbonate solutions were indistinguishable.

1. Introduction The life cycle of Dictyostelium diswideum as observed and reported by Raper (1935) included: the aggregation stage, in which stationary-phase amoebae collect into multicellular aggregates; the pseudoplasmodium stage, in which the conical aggregate

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is transformed into a migrating slug-shaped structure whose movement is controlled by its apical tip, and which senses and responds to horizontal, unidirectional light and to temperature gradients (Raper, 1940: Bonner et al., 1950; Poff et al., 1973; Poff $ Loomis, 1973); the culmination stage. in which the migrating slug stops and constructs a fruiting body. A later study (Newell et al., 1969) revealed that slug migration and fruiting-body construction represent alternative rather than obligatorily sequential developmental programs. Confirming and extending earlier st.udies (Raper, 1940 ; Slifkin & Bonner, 1952), these workers specified alternative sets of environmental conditions (pH, salt concentration, humidity, light) in which newly formed aggregates could be either induced to construct fruiting bodies directly at t,he site of aggregation or transformed into migrating slugs. The slugs remained in that condition indefinitely, hut if shifted to the environment which permitted fruiting, they immediately stopped migrating and constructed fruiting bodies over a seven-hour period regardless of how long they had remained as slugs. Patterns of protein accumulation and disappearance were shown to be profoundly affected by the choice of these morphogenetic a,lternatives and the primary control of these events appeared to be at the level of transcription. It was postulated that some kind of “morphogenetic feedback” operates to inform t,he individual cells of the morphogenetic status of the entire cell assembly a.nd to modulate the programs of gene expression accordingly (Newell & Sussman, 1970: Ellingson et al., 1971; Sussman et al., 1975). The experiments of h’ewell et al. (1969) a 1,so indicated that the transformation of the newly formed aggregate into a migrating slug rather than a fruiting body depended on the local accumulation of a diffusible metabolite excreted before and during aggregation. The subsequent shift back to the fruiting mode could be accounted for by the disappearance or removal of the metabolite. The data summarized in this paper confirm the earlier conclusion and indicate that the metabolite responsible is ammonia.

2. Materials and Methods (a) Organism D. discoideum NC-4 (haploid) was grown in association with Aerobacter aerogenes (Sussman, 1966). Stationary-phase cells harvested from 24-h plates incubated at 22°C were washed 3 times by centrifugation ir, cold water and suspended in a buffered salt solution (LPS) containing: KC1 (1.5 mg/ml) ; MgCl, .6H,O (0.5) ; streptomycin sulfate (0.5) ; 40 rnM-sodium/potassium phosphate (pH 6.5).

(b) Incubation

during

morphogenesis

Samples of 0.5 ml containing 1 x lo7 cells were pipetted onto 2-in Whatman no. 50 filter paper circles. The filters rested on BO-mm Petri dishes on absorbent pads saturated with LPS buffer. Alternatively they were placed in Lucite incubation chambers, each consisting of a lid and reservoir clamped together with a rubber gasket ring between them and, resting on the gasket, a square of dialysis membrane. A filter is placed on the membrane so as to remain in contact with fluid within the reservoir. The reservoir volume can be adjusted between 4 and 30 ml by addition of Lucite blocks. Exit and entry ports permit the reservoir fluid to be sampled and to bo flushed out and replaced whenever desired.

(c) Ammonia Ammonia

was determined

recording spectrophotometer.

enzymatically

determinations by the method

of Tabor

(1966) using a Gilford

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(d) Reagents Crystalline L-glutamate dehydrogenase and reduced (NADH) were purchased from Sigma Corp. [35S]Methionine from New England Nuclear.

diphosphopyridine (420 Ci/mmol)

nucleotide was obtained

3. Results The conclusion that NH, induces newly formed aggregates to transform into migrating slugs, and that its removal permits the slugs to abandon the slug mode and to construct fruiting bodies, is based on the following experimental evidence. (;t) Prior

to and during aggregation the cells produce and excrete a signi$cant quantity of NH,

Washed stationary-phase cells were dispensed on filter paper circles and incubated in Lucite chambers (see Materials and Methods) over reservoirs containing either 4 ml of water or 4 ml of buffered salt solution (LPS). Under both conditions, aggregates were formed with normal timing but in the former, the aggregates transformed into migrating slugs and the pH in the reservoir rose from 6.5 to 7.5, while in the latter they constructed fruiting bodies directly at the site of aggregation without ever assuming the slug shape and the reservoir pH remained below 6.9. At intervals, fluid from duplicate reservoirs was collected and assayed enzymat’ically for total NH, + NH: (*ontent (Tabor. 1966). As seen in Figure 1, under both conditions NH, -+ NH;

Fro. 1. Kinetics of extracellular NH, + NH: accumulation prior to and during aggregation. Samples of 3 x 10s washed, stationery-phase cells were dispensed on filters and incubated in Lucite chambers over reservoirs containing 4 ml of water ( 0) or LPS buffer (a). The morphogenetic sequences are described in the text. The concentrations of NH, + NH: in duplicate reservoir fluids were measured as NH, by the method of Tabor (1966).

accumulated to a plateau concentration of 12 mMt. This is equivalent to a production of 16 pmol/108 cells. Given that Dictyostelium cell protein contains about 15% NH,, this would be consistent with a net loss of 2 mg total protein by degradation during the period in question. In fact, protein content has been reported to decrease from about 5 mg/108 cell to about 3 mg/108 over the same period (White & Sussman, 1961). t The volume of reservoir fluid did not affect the rate of NH, amount over the range 4 to 16 ml.

+ NHj

production

or the total

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(b) Aggregates involved in fruiting-body construction can be induced to abandon that mode and to transform into migrating slugs in the presence of extracellular metabolite(x) previously accumulated by cells that heed aggregated and transformed into migrating slugst. The slug-inducing activity is correlated with the concentration of ammonia and can be destroyed by removing ammonia enzymatically Samples of 2.5 x lo7 cells at the 15-hour stage of development on quarter filters were each shifted to a Lucite chamber containing 4 ml of fluid adjusted to pH 7.3 with 20 mM-sodium/potassium phosphate. When the reservoir contained water, this smaller number of aggregates freed of previously excreted metabolites continued to construct fruiting bodies directly without ever transforming into migrating slugs (Table 1, column 1). In contrast, when the reservoir contained 4 ml of water over which 3 x lo8 amoebae had previously aggregated and formed slugs, the test aggregates themselves developed into slugs (column 2). However, after six to seven hours they reverted to the fruiting mode and proceeded to construct fruits at the normal rate. Aggregates transferred at the 12-hour stage of development not only transformed into slugs but remained in that mode indefinitely. Zero-time cells incubated in the presence of the metabolite preparation failed to aggregate. In confirmation of the earlier study (Newell et al., 1969), the slug-inducing activity 1

TABLE

Transformation

I

Untreated controls

of cell aggregates into migrating slugs induced by extracellular metabolite preparations and by (NH,),CO, 2

3

Crude metobollte prwaratlons

DIstIllate from 2

(10) t 18 & 00 ---l-

‘36 H,tNH~]M otl5h

Residue from 2

5 DIstillate after enzyme treatment

6 Dlstlllate after further enzyme treatment

7 Distillate after incubat!on wth bolted en*yllle

8

Ammomum carbonate

IO

9

Ammomum carbonate

Ammomum corbonote

T--l(17)

‘24

4

-

&

I H -

13.2

24.8

8

21.5

26.3

21.3

The reservoir contained 4-ml vol. of fluids prepared as described in the text and all adjusted to pH 7.3 with 20 maa-potassium/sodium phosphate. The morphogenetic stages attained at 18, 24, and 36 h are schematically illustrated (t r8 etc.). The numbers in parentheses in the boxes refer to the times required by control aggregates to reach the corresponding stages. [NH, + NH:] concentrations listed at the bottom of each column were measured by the method of Tebor (1966) at the time of transfer. t Preliminary experiments aimed at devising a satisfactory assay system for slug induction, and fractionating, and characterizing diffusible metabolite preparations were performed at the Hebrew University in collaboration with Drs Edith Zylber and Amos Cohen.

14

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was found to be volatile at alkaline pH values but not at acid ones. When the metabolite preparation at pH 7.5 was distilled at 100°C until one-third of the original volume had been volatilized and collected, all of the slug-inducing activity was found in the distillate, none in the residue (Table 1, columns 3 to 4). Fifteen-hour aggregates exposed to the undiluted distillate transformed into slugs and remained in that mode indefinitely. The slug-inducing capacity of the metabolite preparations could also be concentrated severalfold (as indicated from sequential dilution assays) by freeze-drying bo 20% of the initial volume. The activity was found to travel through a Sephadex G15 column eluted with water at a rate consistent, with a molecular weight of less than 200. The slug-inducing activity could be reduced or eliminated from the distillate by enzymatic removal of ammonia (Table 1, columns 5 to 7). 4-ml samples of the distillate were incubated for 30 minutes (column 5) or 65 minutes (column 6) with a reaction mixture including 60 units of L-glutamate dehydrogenase (Sigma, crystalline grade) plus 25 mM(fina1 concn)-m-ketoglutarate and 25 mM-NADH that catalyzes the reaction: NH, -{- a-ketoglutarate + NADH + L-glutamate + NAD+ + H,O. Incubation with boiled enzyme was ineffective. The activity of all these metabolite preparations could be progressively reduced by decreasing the pH over the range from 7.8 to 7.0 and was eliminated below pH 7. That the pH changes per se or the variations in ion concentration that accompanied the pH changes were not effective was shown by t,he response to control solutions containing 20 mM-phosphate f 100 mM-NaCl over the entire pH range 6 to 7.5 (Table 2). TABLE 2 Inrlucws

and non-inducers of the transformation of aggregates into migrating under the assay conditions described in Table I Inducers

slurs

Non-inducers

Met,abolite preparations containing more t,han A 10 mix-NH, + NH,+ & 100 m~-NaCl 20 mw-Na/K phosphate at pH 7 to 7.5

Equivalent

NH,OH, NH&l?, (NH,),SO,t WH,),CO,, containing more than 10 mu-(NH, + NH:) :I: 100 mM-NaCl k 20 miw-Na/K phosphate, pH 7 to 7.5

Equivalent solutions adjusted to pH 6 to 6.9. 20 mw-phosphate + 100 mM-NaCl (pH 6 to 74. 20 miw-Na glutamate, glutamine or urea * 20 mM-phosphate (pH 7.3)

t ‘I%(? slugs induced

by these reagents

(c) The slug-inducing

activity

were slightly

shorter

solutions

with

of metabolite preparations

adjusted

less-elongated

to pH

apical

can be mimicked

6 to 6.9.

tips.

by

equiwwlur NH, solutions at equivalent pH values Table 1, columns 8 to 10, summarizes data for (NH&CO, solutions. NH,OH solutions gave precisely the same results and (NH&SO, and NH&l did the same except for minor changes in slug shape. (The slugs were plump and with less welldefined apical tips.) The pH dependence of ammonium carbonate solutions and metabolite preparations was identical over the range 6.5 to 7.5. Ammonium carbonate added to crude metabolite preparations co-separated with the endogenous inducing

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activity through freeze-drying, chromatography on a G15 Sephadex column and distillation at 100°C. The slug-inducing activity disappeared during incubation with reaction mixtures containing L-glutamate dehydrogenase at the same rate as in metabolite preparations containing equivalent concentrations of NH, + NH:. The sensitivity to ammonium carbonate of zero-time cells and those at the 12-hour stage of aggregation was also precisely the same as had been observed with metabolite preparations containing equivalent concentrations of NH, + NH:. Urea, glutamic acid and glutamine were completely inactive at concentrations over the threshold for ammonium carbonate activity. (d) Isolated auto-induced slugs can be made to stop migrating and begin fruit construction by localized enzymatic removal of NH,? It might be argued that the induction and maintenance of the slug mode amongst aggregates formed by cells at high population density, though regulated by NH, concentration, is a laboratory artifact and is not the means by which isolated, selfinduced slugs remain in that condition. To test this possibility, isolated slugs were produced under conditions previously described (Newell & Sussman, 1970) and allowed to migrate over agar toward a horizontal point source of light. Each slug was treated with 0.5 ~1 of a reaction mixture containing 0.1 unit of purified L-glutamate dehydrogenase, 500 mM-cc-ketoglutarate, 1.5 mM-NADH and 500 mM-imidazole buffer (pH 7.25). Controls included distilled water, a mixture containing boiled enzyme, and mixtures from which a-ketog1utarat.e or NADH was omitted. The results are shown in Table 3. Application of the active react,ion mixture caused immediate cessation of migration and construction of a fruiting body whereas almost all the slugs treated with any of the control solutions (or not treated at all) continued to migrate unimpeded. TABLE 3 Treatment

No. slugs treated

No. that

“8 26 “!J 39 26 29

Complete mixture -NADH --cc-ketoglutaratc -Enzyme Mix with boiled enzyme Water

26 2 2 4 3 2

Isolated slugs migrating toward a horizontal point source of light 0.6 ~1 of a solution containing 1.5 mM-NADH, 500 mivf-cc-ketoglutarate, 7.25) and 0.1 unit L-glutamate dehydrogenase, and were then reincubated

(e) Morphology

and physiological

Slugs induced over reservoirs containing monium carbonate were somewhat less oriented with their apical tip pointing away to it. Migration in the dark was minimal ammonia gradient such as a self-induced t It is a pleasure to acknowledge experiment w part of a graduate

the expert introductory

fruited

were sprayed each 500 mix-imidazole 8 h before scoring.

state of NH,-induced

with (pH

slugs

either metabolite preparations or amelongated than auto-induced slugs and from the substratum rather than applied due, we presume, to the absence of an slug might produce. However, they did

assistance research

of MS Michel course.

Sanders

who performed

this

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migrate over considerable distances toward a horizontal point-source of light while in the presence of homogeneously distributed NH,. Anytime that the (NH&CO, solution was removed by flushing the reservoirs with disMled water (even after 48 h) the shift to the fruiting mode was virtually immediate and fruiting-body construction was completed over a seven-hour period, just as observed with auto-induced slugs (Newell et al., 1969). To determine rates of amino a,cid incorporation into protein, quarter filters each bearing 2,5x lo7 cells at the 15-hour stage were transferred t)o chambers over reservoirs containing either water or 22 mM-ammonium carbonate adjusted to pH 7.3 with 20 mM-potassium phosphate. Between 15 and 23 hours, duplicate filters were pulsed for 2-hour periods, with 25 $Zi [3”S]methionine delivered to each reservoir. The incorporat’ion of radioactivity into a,cid-insoluble material was completely unaffected by t’he presence or absence of ammonium carbonate. As seen in Figure 2, the patt#erns of enzyme accumulation and disappearance closely resembled those that have been reported previously for autoinduced slugs (Newell & Sussman, 1970; Ellingson et al., 1971). The sole difference was the cessation in the induced slugs of the accumulation of UDP-glucose p.yrophosphorylase activity rather than merely a retardation of rate. However, when the (NH,),CO,-induced slugs were allowed to fruit by removal of the agent, they synthesized a second quantum of UDP-glucose pyrophosphorylase just as do their autoinduced count’erparts (Newell & Sussman, 1970). The ammonia-induced patterns for

600

-400

-200

a F \ VI f 57 .a E 2 c lL

;4 Time 4 -8

12

16

24

28

Time i h ! Fro. 2. Patterns of enzyme accumulation and disappearance in cell aggregates during exposure to (NH,),CO, and after its removal. At the times designated by the arrows, filters were transferred to chambers over reservoirs containing 27 mix-(NH&CO3 adjusted to pH 7.3 with 20 mwpotassium’phosphate or the latter was removed by flushing the reservoirs with water. At intervals filters were harvested and the enzyme specific activities were measured in the cell extracts by procedures described elsewhere (Newell & Sussman, 1970; Ellingson et nl., 1971). (-O-O-) Cells incubated over reservoirs cont,aining LPS buffer; these constructed fruiting bodies directly.

168

UDP-galactose to be identical

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epimerase and UDP-galactose polysaccharide transferase appeared to those reported for auto-induced slugs (Ellingson et al., 1971).

4. Discussion For D. discoideum amoebae growing either upon a bacterial associate or in axenic medium (Sussman & Sussman, 1967) protein degradation is the primary and perhaps sole source of carbon and energy, and NH, + NH: are major catabolic products. This metabolic pattern is continued during morphogenesis via the degradation of endogenous protein (Gregg et al., 1954; White & Sussman, 1961; Hames & Ashworth, 1974). The data reported here indicate that the resulting accumulation of NH, in the microenvironment induces the newly formed aggregate to transform into a migrating slug and to move away from the site of aggregation. The slug sheath, minimal at the apical tip and progressively thickened posteriorly as cortical cells deposit sheath material, is known to be an effective hindrance to diffusion (Farnsworth & Loomis, 1974). It may well play a major role in maintaining a high level of NH, within the microenvironment, thereby ensuring continuance of the cell assembly in the slug mode. The disappearance of NH,, by efficient diffusion into the substratum, evaporation into the atmosphere, conversion to NH: by a decrease in ambient pH, and decrease in the rate of NH, production with the passage of time, or by experimentally contrived enzymatic removal, immediately induces the slug to stop migrating and enter the fruiting mode. Morphogenetic effects of NH, have been described by previous workers. Cohen (1953) reported that NH,, if present from zero-time, inhibits aggregation; a finding confirmed by Newell et al. (1969) and in the present study. Feit (1969) observed that in a number of species of Dictyosteliaceae, exposure to ammonia increases the sizes Lonski (1976) published of the aggregates and reduces their number. Recently, observations confirming the above and indicating that microcyst formation in P. pallidurn as an alternative to fruit construction can be induced by ammonia. Aggregate size and number were affected by ammonia in a dwarf mutant of D. discoideum but not in the wild type. No other effects were noted. Loomis (1970) reported that exposure of fruiting bodies at the penultimate stage of spore differentiation to atmospheric NH, prevented further development. In the earlier study of Newell et al. (1969), the notion that NH, might be the inducer of slug formation was entertained but was discarded primarily because concentrat.ions of NH,OH present from zero-time which were low enough to permit aggregation failed to influence the course of fruit construction. Exposure of already aggregated cells under conditions which prevented the dissipation of NH, via evaporation was not attempted. 3’5’ cyclicAMP has also been shown to regulate the course of morphogenesis and gene expression in D. discoideum : in triggering the acquisition of cohesivity (Gerisch et al., 1975) ; in inducing the synthesis of phosphodiesterase (Klein, 1975) ; in possibly polarity and apical directing slug migration (Bonner, 1949) ; and in maintaining dominance in both the migrating slug and the developing fruiting body (Nestle & Sussman, 1972). In order to determine the site of action of NH, as a morphogen it would seem essential first to establish if NH, affects in any way the production and/or release of or response to cyclicAMP. Experiments are underway toward this end. It is possible to construct a model of morphogenetic regulation (Sussman et al., 1977),

MOHPHOGENETIC

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I69

based on the actions of NH, and cyclicAMP and interactions between them, which accounts for the primary aspects of morphogenesis and cytodifferentiation in this organism including the transition between slug migration and fruit construction. The latter emerges as the consequence of two assumptions: (a) that NH, interferes with the synthesis of cyclicAMP or its release or both ; (b) that cyclicAMP serves as a chcmotactic attractant to cells within the aggregat)e just as it does at an earlier time during aggregation itself. This work was supported tion to one of us (M. S.).

by a grant

(PCM76-03993)

from the National

Science

Founda-

REFERENCES Bonner, J. T. (1949).J. ,&pZ. ZooZ. 110, 2599271. Bonner, J. T., Clark, W., Neely, C. & Slifkin, M. (1950). J. CeZZ.Comp. Physiol. 36,149-155. Cohen, A. L. (1953). Proc. Nat. Acad. Sci., U.S.A. 39, 68-74. Ellingson, J. S., Telser, A. & Sussman, M. (1971). Biochim. Biophys. Acta, 244, 388-395. Farnsworth, P. & Loomis, W. F. (1974). Develop. Biol. 41, 77-85. Feit, 1. (1969). Ph.D. thesis, Princeton University, Princeton, New Jersey. Gerisch, G., Fromm, H., Huesgen, A. & Wick, V. (1975). Nature (London), 255,547~549. Gregg, J., Hackney, A. & Krivanek, J. (1954). Biol. BUZZ. 197, 226-235. Hames, B. D. & Ashworth, J. M. (1974). Biochem. J. 142, 301-315. Klein, C. (1975). J. BioZ. Chem. 250, 7134-7138. Lonski, J. (1976). Develop. BioZ. 51, 158-165. Loomis, W. F. (1970). Ezpt. Cell Res. 60, 2855291. Nestle, M. & Sussman, M. (1972). Develop. BioZ. 28, 545-554. Newell, P. C. & Sussman, M. (1970). J. ikfol. Biol. 49, 627-637. 100, 763-768. Newell, P. C., Tclser, A. & Sussman, M. (1969). J. Bacterial. Poff, K. B. & Loomis, TV. F. (1973). Ezpt. Cell Res. 82, 236-240. Poff, K. I,., Butler, W. L. & Loomis, W. F. (1973). I’TOC. Nat. Acad. Sci., U.S.A. 70, 813 Raper, K. B. (1935). J. Agric. Res. 50, 135-147. Raper, I<. R. (1940). J. Elisha Mitchell Sci. Sot. 56, 241- 282. Slifkin, M. dz Bonner, J. T. (1952). BioZ. Bull. 102, 273-277. Sussman, M. (1966). Methods in Cell Physiology (Prescott, D., ed.), pp. 397-410, Academic Press, New York. Sussman, M., Alexander, S., Boschwitz, C., Brackenbury, R. W., Cohen, A. BESchindler, J. ( 1975). ICAr- UCLA Winter Conference on Developmental Biology (MacMahon, D. & Fox, C. F., eds), pp. 89-108, W. Benjamin Pub., Philadelphia. Sussman, M., Schindler, J. di Kim, H. (1977). Proc. EMBO Workshop on Develop. and Differentiation in Cellular Slime Molds (Capuccinrlli, P., ed.), Elsevier Press, Amsterdam, in the press. Sussman, R. It. & Sussman, M. (1967). Biochem. Biophys. Res. Com.mun. 29, 53-55. Tahor, C. W. (1966). Methods in Enzymology, vol. 17A, p. 955, Academic Press, New York. Whit’e. G. ?J. & Hussman, M. (1961). Riochim. Rioph,ys. Acta, 53, 285-293.