B, tetrazoles, and the Nadi reagent.1 Interpretation of the results obtained with

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1 ENZ YML' LOCALIZA 7ON IN AZOTOBACTER VINELA ND)II* By MARTIN ALEXANDERt AND P. W. WILSON DEPARTMENT OF BACTERIOLOGY, UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN Communicated July 8, 1955 INTRODUCTION Although a considerable literature exists on the localization of enzymes in the cells of multicellular organisms, little information is available dealing with the correlation of the microbial cell structure with its physiology. Most studies of the metabolic properties of microbial structures such as granules have been cytochemical-for example, using such oxidation-reduction indicators as Janus Green B, tetrazoles, and the Nadi reagent.1 Interpretation of the results obtained with such methods has led to considerable controversy.2 A more objective method is that used so extensively in studies with mammalian liver cells-namely, a direct determination of the chemical and physiological properties of structures separated and isolated by differential centrifugation of cell extracts. This technique has the advantage not only of being applicable to structures too small to be resolved in the light microscope but also of being adaptable to many enzymes with the specific enzymatic reaction being determined rather than some secondary, indicator reaction. Slonimsky and Hirsch3 employed this method to differentiate normal and "petite" strains of Saccharomyces cerevisiae, and Gunsalus, Gunsalus, and Stanier4 also used it to localize several enzymes in the mandelic acid metabolism of Pseudomonas fluorescens. The research described in this report illustrates the usefulness of this tool, particularly when it is sharpened to include quantitative studies that enable the investigator to draw up a balance sheet for all the cellular material and all the enzymatic activity. Failure to do this may lead to confusing, contradictory, and even erroneous conclusions. CRITERIA FOR LOCALIZATION To overcome the confusion that results from the use of qualitative data in cytochemical studies, it is necessary to set up criteria for determining the site of activity of an enzyme and for serving as standards of cytochemical significance. Initially, a balance sheet accounting for all the cellular mass and all the enzymic activity associated with it must be set up. If the intracellular distribution of some substance is then to be ascertained, the amount of that substance recovered following fractionation must be essentially equal to that in the original cell extract. Low recoveries of an enzyme preclude a definite conclusion regarding its localization. For the association of a metabolic activity with a particular intracellular body, a large percentage of that activity must reside in the fraction isolated from the cellfree preparation composed of that structure. The presence of a small percentage of the activity in any one fraction can, at best, be of only doubtful cytochemical significance. If two cell components are responsible for a certain function, with one comprising a greater quantity of the protoplasmic material than the other, the low total activity in the less abundant constituent will frequently be overlooked. Such limi- 843

2 844 BACTERIOLOGY: ALEXANDER AND WILSON PROC. N. A. S. tations can be overcome by employing another useful criterion, viz., the concentration of the property in the subcellular unit. If, for example, this concentration standard is defined as the ratio of the specific activity (units of enzyme per milligram of nitrogen) in the fraction to that in the cell extract, then cytochemical significance should be ascribed only to a fraction which possesses a concentration coefficient equal to or greater than that of the unfractionated extract. Three requirements must therefore be satisfied to present valid data relating physiological properties of the cell to its subcellular constituents: (1) recovery of both enzyme and cell mass (nitrogen) in the isolated fractions must approach 100 per cent of that found in the original bacterial extract; (2) a high percentage of the total activity must reside in the fraction to which the function is being attributed; and (3) the concentration of the enzyme in the isolated fraction must be equal to or greater than that in the initial cell-free preparation. EXPERIMENTAL Methods.-Sixty grams of a washed cell paste of Azotobacter vinelandii 0 harvested from a 24-hour culture grown in nitrogen-free mediums are ground with 10 gm. of levigated alumina and the mixture extracted with 100 ml. of a cold buffered lactose solution (0.15 M C.P. lactose in 0.02 M potassium phosphate buffer, ph 7.0). The alumina is removed by centrifugation at 650 X g for 15 minutes (centrifugal forces reported in this paper are the forces prevailing at the bottom of the centrifuge tube). All the material remaining above the white abrasive precipitate is recentrifuged at 2,000 X g for 20 minutes in a refrigerated centrifuge; the clearer top layer is removed first, then the lower pink layer is carefully separated from the bottom tan sediment of cells. To the combined pink layers is added buffered lactose, and the suspension is resedimented at 2,000 X g for 20 minutes. The pink material is again separated from the lower tan sediment, combined with the initial supernatant, and brought to 100 ml. with cold buffered lactose solution. This is designated the cell extract, "CE." A 75-ml. aliquot is removed and subjected to a centrifugal force of 7,000 X g for 30 minutes, and the residue is washed with a small amount of 0.15 M lactose and resedimented at 25,000 X g for 30 minutes. The particulate material is brought into a homogeneous suspension with a hand-model tissue grinder, diluted to 25 ml. with buffered lactose, and designated the largeparticle fraction, "25p3O."P6 The combined supernatants are centrifuged at 60,000 X g for 60 minutes; the pellet, suspended by a brief homogenization and brought to a 10.0-ml. volume with the lactose menstruum, is termed particulate fraction "60p60." Fluffy material above the residue or material rising centripetally in the centrifuge tube is always kept with the nonsedimented components. After the supernatant is recentrifuged for 60 minutes at 144,000 X g, the precipitate is suspended in a 10.0-ml. volume and labeled the "144p60" particle fraction. To bring out residual granular components, KCl is added to a final concentration of 0.3 M, and the 144pK30 fraction sedimented at 144,000 X g for 30 minutes. The volume of the final supernatant ("S" fraction) is accurately measured. The supernatant fraction is presumed to correspond to the "ground cytoplasm"-i.e., the nonparticulate components of the cell. It is termed "soluble" in an operational rather than inma-physicochemical sense. Thus a "soluble" enzyme is one which cannot be sedimented by the maximum centrifugal forces employed.

3 VOL. 41, 1955 BACTERIOLOGY: ALEXANDER AND WILSON 845 For the localization of alkaline phosphatase, the extracting fluid consisted of 0.15M C.P. lactose-o.05m KCl. Succinoxidase activity was determined by the method described by Repaske,7 adenosine deaminase by a spectrophotometric technique,8 and alkaline phosphatase by the liberation of orthophosphate.9 Catalase was determined manometrically,10 using the rate of 02 evolution in the first 2 minutes. General Properties.-The small particles (6Op6O, 144p60, 144pK30) sedimented during ultracentrifugation form a red gel-like pellet which is suspended only with difficulty. These fractions account for 15.3 per cent of the nitrogen of the cell extract (CE). The large-particle fraction (25p30) contributes 15.5 per cent of the total nitrogen and appears in the undisturbed centrifuge tube as a pink, loose layer sedimentable at low speeds. The rest of the cellular mass (66 per cent) is found in the clear, yellow supernatant fraction (S). The distribution of nucleic acids in the fractions is shown in Table 1. Pentose nucleic acid is found in both particulate and nonsedimentable components, its distribution paralleling that of the nitrogen. The similarities in concentrations among the various fractions indicates that there is no single site of PNA localization. Deoxypentose nucleic acid apparently is completely in the soluble ground plasm, with 92.5 per cent of the initial DNA found in that fraction alone. These data indicate that none of the particles separated in these studies represents an isolated nucleus. Such recovery of the DNA with the nonsedimentable constituents is a peculiarity neither of A. vinelandii nor of the method of cell breakage, since similar results have been reported with numerous microorganisms with several different methods of disrupting the cell surface. TABLE 1 INTRACELLULAR LOCALIZATION OF DEOXYPENTOSE NUCLEIC ACID (DNA) AND PENTOSE NUCLEIC ACID (PNA) DNA- - PNA - FRACTION Per Cent CE Concentration Per Cent CE Concentration CE (100) (1.0) (100) (1.0) 25p3O Op6O p6O pK S Per cent recovery The finding of nuclear material with the nonparticulate cell constituents may not be a true reflection of the cell structure, since, in animal tissues at least, the nuclear membrane is fragile and may be ruptured by procedures used to break the cell. Alternatively, the bacterial nucleus may be organized in some more primitive form-for example, lacking a limiting membrane. Regardless of the explanation, it is evident that the bacterial DNA is not tightly bound to any granular structure. Since this compound is effectively soluble in azotobacter extracts, its presence in particle fractions may be used as a test of the degree of contamination of the granules with the supernatant material. Photomicrographs of the 25p3O fraction reveal large, electron-dense structures varying in shape from oval to spherical. Direct measurements frbm the micrographs give a size range for these granules of ,u in diameter, while estimation of diameter by the rate of sedimentation indicates particles of the order of 0.4,u.

4 846 BACTERIOLOGY: ALEXANDER AND WILSON PROC. N. A. S. These dimensions agree well with the size of the microscopically discernible granules in the intact azotobacter cell as well as in other bacteria. The submicroscopic granules of A. vinelandii are spherical in shape and cast a considerable shadow; their range in diameter is 15-25m~t. Studies of their rate of sedimentation in the analytical centrifuge indicate the presence of two separate structures having diameters of 19 and 15 m~i. If some other method of breakage was used to avoid the alumina, a white fraction was recovered which sedimented at a faster rate than the 25p30 particles. Upon isolation and purification, these structures were shown to be isolated cell walls. Because of the difficulties in achieving complete separation of walls from residual cells without exhaustive and time-consuming procedures, the wall layer was not included in any of these localization studies, although its enzymatic properties should prove interesting. Localization of Enzymes.-The intracellular distribution of succinoxidase, which catalyzes the aerobic oxidation of succinic acid, converting it to fumarate, is given in Table 2. Recovery of the activity of the extract is high, 92.7 per cent being found in the isolated fractions. The enzymatic activity is clearly a function of the cell granules as judged by both the percentage and the concentration criterion. More than 85 per cent of the activity recovered was found in the particles, the concentration being six to thirty times greater in these than in the supernatant fractions. Significantly, most of the granule-bound activity is found with the smaller structures, only about one-fifth being in the 25p3O fraction. Even this activity may be a result of the occlusion of submicroscopic bodies, since such structures were always seen in electron photomicrographs of this fraction. It appears that succinoxidase is associated with the granular components of the azotobacter cell and primarily if not exclusively with the constituents of size mpc. TABLE 2 SUCCINOXIDASE DISTRIBUTION IN FRACTIONS OF Azotobacter vinelandii* Per Cent Units/ Fraction Units CE Mg N Concentration CE 70,900 (100) 171 (1.0) 25p30 11, p60 11, p60 18, pK30 15, S 9, Per cent recovery * Assay: 16p M histidine, ph 6.5; 5 pm Mg5O4; 50 pm succinate; KOH in center well; temp., 330C. 1 unit of succinoxidase: the amount of enzyme which utilizes 1,1 02/br. Adenosine deaminase catalyzes the conversion of adenosine to inosine with the liberation of ammonia. It probably functions in the interconversion or in the decomposition of these metabolically important nucleosides. The enzyme from A. vinelandii forms inosine from adenosine in almost quantitative yields. The deaminase is exclusively found in the supernatant of the cell-free extract (Table 3). The high recovery in the S fraction eliminated the need for further characterization of the particles. The concentration coefficient in this single fraction was An identical distribution of this enzyme has been reported in cells of animal tissue.8

5 VOL. 41, 1955 BACTERIOLOGY: ALEXANDER AND WILSON 847 As shown in Table 4, an entirely different type of distribution is found for catalase. On the basis solely of the percentage criterion, it would appear that this enzyme is a property of the nonsedimentable portion of the cell constituents. But, since the concentrations in all fractions are similar to that of CE, i.e., about 1.0, it is evident that catalase is found in all parts of the bacterium in proportion to the TABLE 3 SITE OF AcTIVITY OF ADENOSINE DEAMINASE* Units/ Fraction Units Per Cent CE Mg N Concentration CE 58,900 (100) 186 (1.0) S 55, Per cent recovery * Assay: 12 pm histidine, ph 7.0; 0.2 pm adenosine; enzyme; water to 3.0 ml. 1 unit of adenosine deaminase: the amount of enzyme to give a change in OD266 of per minute. nitrogen content of the structures involved. It is doubtful that absorption phenomena account for these data because of the high enzyme concentration in the particles. In support of the view that the H202-decomposing activity is in part granular are the observations that purified 40 S (Svedberg units) particles contain catalase." TABLE 4 INTRACELLULAR LOCALIZATION OF CATALASE AND ALKALINE PHOSPHATASE CATALASE - - -ALKALINE PHOSPHATAsEt-- FRACTION Per Cent CE Concentration Per Cent CE Concentration CE (100) (1.0) (100) (1.0) 25p p p pK S Per cent recovery * Assay: 441 FM H202; pm phosphate, ph 7.0; KOH in center well; temp., 300 C. t Assay: 100 pm P glycerophosphate; 200 pm veronal buffer, ph 9.5; 5 AM MgCl2; enzyme; water to 5.0 mmml. Determine orthophosphate after 15 minutes' incubation at 38 C. Of especial significance to these studies is the intracellular localization of the alkaline phosphatase, since the latter is one of the few enzymes localized in bacteria by direct microscopic observation of a cytochemical reaction.12 Alkaline phosphatase is clearly associated with the soluble cell material; the slight activity in the granules probably is of no cytochemical significance (Table 4). These biochemical data are in contradiction to the cytological results of Schaechter et al.,12 who employed the Gomori method; the use of this method for enzyme localization, however, has been criticized. 13 SUMMARY AND CONCLUSIONS The intracellular localization of several enzymes has been studied in A. vinelandii 0 by isolation of the cellular constituents in a buffered lactose menstruum. Criteria have been discussed by which valid enzyme distribution in the bacterial cell can be ascertained. The succinoxidase system of the azotobacter is localized in the particles, with the greatest activity being associated with the submicroscopic

6 848 BACTERIOLOGY: ALEXANDER AND WILSON PROC. N. A. S. granules. Adenosine deaminase and alkaline phosphatase are found in the nonparticulate components of the cell, whereas catalase appears to be associated with both granular and soluble cell material. The distribution results indicate a marked similarity of the organization of the microorganism to that of the cells of higher plants and animals, although the presence of a large granule which gives a positive metachromatic reaction for polymetaphosphate may indicate the presence of an energy-storage structure within the microbial cell. The absence of a nuclear particle in these bacterial extracts leads to some problems with regard to cytochemical studies. Although the "soluble" fraction (S) represents to a great degree the ground substance of the cell, the presence of DNA in that fraction can result in erroneous conclusions. If there does exist a nuclear particle in bacteria, enzymes bound to it should be recovered in the DNAcontaining fraction, namely S. It is still necessary, then, to find a means of isolating the bacterial nucleus free of the cell, so that its biochemical properties may be examined directly. Its small size has made the bacterial cell difficult for the cytologist to study, but, as is illustrated in this paper, the role of the subcellular structures in the physiology of the organism can be simply and directly determined on structures isolated from ruptured cells. Nevertheless, there remains a definite need for microscopic studies in enzymatic cytochemistry, since biochemical techniques give no indication of the position and arrangement in the intact organism of the isolated structures. * This research was supported in part by grants from the Rockefeller Foundation and the Atomic Energy Commission. t National Science Foundation Fellow. Present address: Department of Agronomy, Cornell University, Ithaca, New York. I S. Mudd, L. C. Winterscheid, E. D. DeLamater, and L. J. Henderson, J. Bacteriol., 62, 459, C. Weibull, J. Bacteriol., 66, 137, 1953; E. A. Grula and S. E. Hartsell, J. Bacteriol., 68, 498, P. P. Slonimsky and H. M. Hirsch, Compt. rend. Acad. sci, (Paris), 235, 741, I. C. Gunsalus, C. F. Gunsalus, and R. Y. Stanier, J. Bacteriol., 66, 538, P. W. Wilson and S. G. Knight, Experiments in Bacterial Physiology (Minneapolis: Burgess Publishing Co., 1952). 6 The symbols for the particles indicate the centrifugal force in 1,000 X g and the time in minutes necessaty to sediment the particle, "p." Although somewhat cumbersome, it seems more useful than the simpler but noninformative "A," "B," and "C." 7 R. Repaske, J. Bacteriol., 68, 555, W. C. Schneider and G. H. Hogeboom, J. Biol. Chem., 195, 161, R. K. Morton, Biochem. J., 57, 595, N. L. Lawrence and H. A. Halvorson, J. Bacteriol., 68, 334, "1 H. A. Schachman, A. B. Pardee, and R. Y. Stanier, Arch. Biochem. and Biophys., 38, 245, 1952; A. F. Brodie and F. Lipmann, J. Biol. Chem., 212, 677, M. Schaechter, E. L. Treece, and E. D. DeLamater, Exptl. Cell Research, 6, 361, A. B. Novilooff, Science, 113, 320, 1951.