Principles of Tissue Fractionation

1. Theoret. Biol. (1964) 6, 33-59

CHRISTIAN DE DUVE Laboratory of Physiological Chemistry University of Louvain, Belgium and The Rockefeiler Institute, New York, N. Y., U.S.A.

(Received 15 April 1963)

This paper representsan attempt to develop logically the basic premises that tissuefractionation is: (a) a chemical method to be conducted according to the ground rules which govern chemical fractionation in general; (b) potentially applicable to the separationand characterization of all elements of cellular organization, whether known or unknown, which are not irretrievably lost in the initial grinding of the cells. It is shownthat the approach which best answersthese prerequisites is a purely analytical one, untrammeled by any preconceivedidea of the cytological composition of the isolated fractions, and allowing their bio- chemicalproperties to be expressedas continuous functions of the physical parameterwhich determinesthe behaviour of subcellularcomponents in the fractionation systemchosen, Examples are given which illustrate the application of density gradient centrifugation in this type of approach, as well as the advantageswhich can be derived from the useof mediaof different composition. The resultsof suchexperiments are expressedin the form of distribution curves of biochemicalconstituents, as with other fractionation methods such as chromatographyor electrophoresis,but with the differencethat the independentvariable is related to a property, not of the constituent but of its host-particles. These curves can be taken to representthe mass distribution of the particles themselvesif the constituent is assumedto be homogeneouslydistributed amongstthem (postulateof biochemicalhomo- geneity). This assumptionhas been verified for a number of enzymes,which provide valuable markersto fix the position of their host-particleson the distribution diagrams. By comparingthe distribution of an unlocalizedconstituent againstthe background of known distributions, especiallyunder a variety of experimental conditions, and by making use of all additional data which can be obtained by ancillary experiments,it is usually possibleeither to demonstrate the associationof the constituent with a known intracellular component, or to bring to light its possiblelocalization in an as yet unidentified type of particle. The existence,chemical properties, and structural features T.B. 33 3 34 CHRISTIAN DE DUVE of the latter can be further established when enough analytical resolution has been achieved to warrant a preparative attempt. Lysosomes as well as microbodies containing urate oxidase, catalase and D-amino acid oxidase have been identified in rat liver by following an approach of this kind. In the design of tissue fractionation experiments and in the interpretation of their results, it is essential to observe a rigorous logic and to employ an appropriately accurate vocabulary. The most important requirement in this respect is to maintain a strict distinction between the intracellular organelles or structures as they occur within the cells, the populations of particulate aggregates as they are present in the homogenate and react to the fractionation procedure, and the subcellular fractions as they are isolated and analysed.

Introduction Numerous reviews have dealt with the theoretical basis and practical applications of centrifugal fractionation. Most recent are that of Allfrey (1959), in which technical procedures are described and discussed in a particularly thorough fashion, and that of de Duve, Berthet & Beaufay (1959) which is specially devoted to density gradient centrifugation and includes a fairly complete mathematical treatment of the behaviour of a particle moving under the influence of centrifugal force in a homogeneous or in a heterogeneous medium. The object of the present paper is somewhat different and represents an attempt to outline what may be termed conceptual aspects of tissue fractionation. The author and his associates began applying this technique shortly after it was first described and have used it more or less continuously ever since. The particular nature of the problems to which they became exposed, together with various results obtained by other investigators, led them to deviate progressively from the approach proposed by the originators of the method. As is often the case, the change was made empirically, largely by trial and error and with no clear understanding of the issues involved. A survey of the recent literature indicates that some workers have undergone a similar evolution, while many others still adhere to the original approach or, more exactly, to a somewhat distorted version of it. Looking back upon these investigations in the light of present knowledge, one can now recognize more easily the logical basis of the new approach as well as the nature of the premises from which it is derived. This paper is largely devoted to a retrospective analysis of this kind. Although written in the form of a personal essay, it is not intended to be dogmatic and is presented essentially in the hope that it will be of help to other workers engaged in tissue fractionation and will stimulate them to reflect more deeply on the significance and potentialities of this remarkable experimental tool. A shorter article on the same subject has already been published (de Duve, 1963). PRINCIPLES OF TISSUE FRACTIONATION 35 In writing this paper, it has been repeatedly necessary to distinguish clearly between two types of intracellular entities : the morphological components or organelles, such as the nuclei, the mitochondria and other intracellular particles, and the biochemical constituents, such as proteins, nucleic acids, enzymes, coenzymes, trace metals, and so on. In order to facilitate this distinction, the word component has been used consistently for the structural entities, and the term constituent for the biochemical ones. In line with this convention, one of the main objects of cytochemistry may be defined as the structural localization of cell constituents, or alternatively, as the biochemical characterization of cell components. Centrifuge and Microscope Cytochemistry Though not originally developed by biochemists, tissue fractionation is fundamentally a chemical method. It may be defined as the chemist’s approach to an integrated study of cell structure and function, as opposed to that of the morphologist, cytochemical visualization. While both methods are directed primarily towards the correlation of biochemical events with subcellular structures, the manner in which this common aim is pursued is charac- teristically influenced in each case by the biases of the parent discipline. In visual cytochemistry, the main concern is to render biochemical constituents and enzymes visible within the familiar framework of tissue or cell architecture. Preservation of the latter is perforce a dominant factor and is achieved at the cost of variable but usually fairly high losses in biochemical precision. On the other hand, fractionation methods follow the classical steps of chemical separation and analysis, and sacrifice a great deal of morphological information to their basic requirements for biochemical integrity and analytical accuracy. Both approaches fall short of the high standards adopted in the more classical forms of biochemistry or cytology, and it is therefore not sur- prising that they have inspired a number of pessimistic pronouncements, usually emanating from one side and directed at the other. In general, the view that an enzyme may emerge unscathed from the usual fixation procedures and will later avoid a number of kinetic traps and conveniently surround itself with the artificially insolubilized products of its own action, is as abhorrent to the enzyme chemist as is to the morphologist the concept that an intact nucleus or mitochondrion may somehow miraculously be separated with a centrfuge from the remains of chaotically dismembered tissues. Such criticisms, though useful in stimulating a constant search for improve- ment, leave out the fact that the standards of biochemistry and cytology have been raised to their present degree of eminence mostly by a process of mutual exclusion, in which structural and biochemical integrity have been deliberately 3--z 36 CHRISTIAN DE DUVE sacrificed to one another. To condemn any correlative approach a priori on the basis of a single set of criteria is actually to deny the possibility of such an approach, which of necessity must rest on a compromise. It is to forget that with all its defects, the correlative attempt must bring the investigator in closer contact with the true properties of the living cell, in which structure and function are undissociable. A more constructive attitude is that adopted by many workers in both fields, who consider the two approaches as comple- mentary rather than opposed, and the artifacts involved as factors to be clearly recognized, minimized by all possible means and taken into account, when unavoidable, in a realistic interpretation of the results. In practice, owing to a fundamental difference between the two techniques, they have so far covered little common ground and rendered unequal services. In visual cytochemistry, especially when directed at the intracellular localization of enzymes, the main limitation lies in the enzymes themselves, of which only a few have so far lent themselves to appropriate staining procedures. In addition, years of arduous work may be necessary to set up optimal conditions and to attain some degree of resolution and specificity. For these reasons the general tendency, in this type of approach, has been to study a restricted number of enzymes on a great variety of experimental material. In contrast, the fractionation procedures allow the study of an almost un- limited number of enzymes and systems, but are not as easily adaptable to different materials. As a result, they have been applied mostly to a few selected tissues but have, in compensation, furnished a considerable amount of biochemical information. At the present juncture it is probably more useful to learn much about few cells than the opposite. Ultimately, however, the last word will rest with the visual cytochemist, since he is best equipped to distinguish individual cells within a tissue and homologous particles within a cell. When comparing the two approaches, it is often assumed that they are both dependent on the state of knowledge in the field which they are trying to invade. The aim of cytochemical staining is implicitly believed to consist in the intracellular localization of known chemical or enzymatic constituents, while that of fractionation methods is taken to be the biochemical characterization of previously identified subcellular entities. This view is not entirely correct. Many staining reactions were discovered empirically without prior knowledge of their object or mechanism, and some of them played an important role in furthering biochemical research. Conversely, the application of tissue fractionation has led in some cases to the discovery of new intracellular organelles, and provided in this manner a considerable impetus to morphological investigation. In conclusion, tissue fractionation must be considered an essentially PRINCIPLES OF TISSUE FRACTIONATION 37 chemical method, comparable to the analogous techniques developed for the separation of proteins or nucleic acids, and differing from them only by the nature of their object and by that of the corresponding separation procedures. As such, it must be conducted according to the ground rules which govern chemical fractionations in general. Moreover, the scope of tissue fractionation is not restricted to the isolation and characterization of a few selected subcellular components. All elements of cellular organization, whether known or unknown, which are not irretrievably lost in the initial grinding of the cells are a valid object for fractionation techniques, which, in fact, provide the most powerful tool presently available for their functional study.

Analytical and Preparative Fractionation When any type of mixture is subjected to a fractionation, this may be done with either an analytical or a preparative aim. Ideally, the two aims are combined and the fractionation then yields a collection of pure fractions separated in a quantitative fashion. In a non-ideal situation, it is often necessary to make a choice between the two objectives and accordingly to sacrifice purity to yield or the reverse. This problem is a familiar one in many areas of chemistry, where analytical procedures of high resolving power are employed to assess the composition of complex mixtures and to evaluate the outcome of the usually less efficient preparative techniques. Tissue fractionation is no exception in this respect and can also be applied either for preparative or for anaIytica1 purposes. However, owing to a number of historical and other reasons, the distinction has not always been clearly made nor driven to all its logical conclusions. The earliest investigations in which fractionation techniques were used were definitely preparative in nature and many of their present applications are still carried out with the same purpose. All the workers who, after Miescher, set out to isolate nuclei, did so with the explicit intention of separating “pure” nuclei, irrespectively of yield (see the reviews by Dounce, 1950; Roodyn, 1959). Again, the pioneering investigations of Bensley & Hoerr (1934) were manifestly directed towards the preparation of mitochondria “in pure form”, a concern which is shared, at least implicitly, by all investigators who routinely isolate mitochondrial fractions for the study of oxidative phosphorylation or of other metabolic functions. In recent years, numerous other attempts at purification have been made and the literature now includes studies on a great variety of nuclear and cytoplasmic inclusions from both plant and animal origin. In all these cases, the emphasis is on the purity of the isolated material, while quantitative recovery is considered of secondary importance. By insisting on the latter requirement and designing a fractionation 38 CHRISTIAN DE DUVE procedure accordingly, Claude (1946a, b) and, after him, Hogeboom, Schneider & Palade (1948) and Schneider (1948) turned cell fractionation into an analytical method. At the’same time, they tried as much as was feasible to combine this new exigency with the preparative aim of the early investigators. Their efforts led directly to the establishment of the now classical scheme, according to which tissues are divided into four fractions, each of which contains, separated as cleanly as possible from the others, one of the major subcellular components of cells : the nuclei, the large granules or mitochondria, the small granules or microsomes, now known to consist largely of fragments of the endoplasmic reticulum with their attached ribosomes, and the cell sap. This is the scheme which is still applied by most workers, except in the case of glandular tissues, from which an additional fraction containing mostly secretory granules is sometimes isolated. Besides its historical associations, there was much to commend the choice of the four-fraction scheme. It was essentially consonant with the rudimentary knowledge of cell structure available at the time, and as is known by anybody who has applied the method, was practically dictated by the macroscopic aspect of the sediments which tend to form layers of distinctly. different color and texture. Additional support to the scheme came from the early biochemical analyses which showed that several enzymatic activities were largely associated with a single fraction, and from the first electron-microscopic observations which clearly confirmed their suspected cytological significance. These features, which characterize what may be termed the pseudo-preparative aspect of the method, have undoubtedly been very helpful in the early development of quantitative tissue fractionation. However, they have a certain compelling quality which has misled many workers into overestimating the preparative value of the technique and has tended to obscure its essentially analytical nature. Such is not the case when the fractionation scheme is entirely arbitrary. The first experiment of this type was carried out by Chantrenne (1947) who studied the distribution of several biochemical properties of cytoplasmic particles as a function of the centrifugal force necessary to bring about their sedimentation. Later, Novikoff, Podber, Ryan & Noe (1953) divided liver homogenates into up to ten fractions by the conventional procedure of differential centrifugation and carried out a correlated study of their properties on the basis of macroscopic and microscopic examination of the pellets, particle-size estimation and enzymic measurements. Somewhat similar experiments were performed by the Louvain group (Appelmans, Wattiaux & de Duve, 1955; de Duve, Pressman, Gianetto, Wattiaux & Appelmans, 1955), except that no morphological examinations were carried out. A more refined analytical procedure, also based on sedimentation rate, was PRINCIPLES OF TISSUE FRACTIONATION 39 introduced by Hogeboom, Kuff and co-workers (Hogeboom & Kuff, 1954; Kuff, Hogeboom & Striebich, 1955; Kuff, Hogeboom & Dalton, 1956) and by Thomson and his group (Thomson & Mikuta, 1954; Thomson & Moss, 1956; Thomson & Klipfel, 1957, 1958). In this procedure, the material is subjected to incomplete sedimentation in a stabilizing density gradient and the distribution of biochemical constituents is measured quantitatively at the end of centrifugation and expressed as a continuous function of radial distance. By further mathematical conversion, the results are finally given in the form of frequency distribution curves relating the measured properties to particle- size or, preferably, to sedimentation coefficient. In another similar application, first used in cell fractionation work by Holter, Ottesen & Weber (1953), the material is brought to virtual density equilibrium with the medium by prolonged centrifugation at high speed in an appropriate gradient and the tube contents are analysed as in the former method. If the density gradient is continuous and accurately known, the results may be used directly to construct frequency distribution curves as a function of another parameter, which is the equilibrium density. The theoretical basis and practical applications of these techniques have been discussed elaborately by Anderson (1955, 1956), de Duve et al. (1959) and Beaufay & Berthet (1963). In judging the relative merits of these various approaches, it is necessary to take into account the composition of the mixtures subjected to fractionation, the resolving power of the applied separation procedures and the sensitivity and accuracy of the methods whereby the composition of the fractions can be assessed. The starting material is generally represented by a “homogenate”, prepared by grinding cell suspensions or, more frequently, solid tissues in an appropriate liquid medium. Even assuming an ideally simple homogenate, i.e. a suspension obtained from a homogeneous cell population without complicating artifacts, one is already dealing with a highly complex mixture mirroring the intricate organization of the original cells. The mixture will be found to contain in solution the constituents of the presumably structureless cell sap, and in suspension a variety of particulate entities forming a number of distinct populations. Amongst the latter, attention may easily be focused primarily on those particles which either by their size or by their number make up a considerable proportion of the total material and may be called major cell components such as, for instance, the nuclei or the mitochondria; but it must be remembered that cells also contain a number of minor components of small size and rare occurrence which, though negligible in terms of bulk, may nevertheless be the bearers of specific and important functions. Several examples of such components are now known and it is likely that others are still awaiting characterization. 40 CHRISTIAN DE DUVE When, as is most frequently the case, several cell types are present in the homogenized material, the complexities described above become multiplied accordingly. In addition, the picture is further distorted by a number of artifacts resulting from mechanical stresses and exposure to unnatural conditions. Most frequently observed are leaking of soluble material from damaged particles or its adsorption onto the surface of particulate elements, fragmen- tation of the latter or their agglutination. The methods presently available for resolving these complex mixtures into their individual components depend largely on differences in such physical properties as size, shape, density and the resulting sedimentation coefficient, to which may be added, in some exceptional cases, electrical charge or solvent affinity. Although they actually offer a wider spectrum of possibilities than is generally suspected, they remain severely limited by the necessity of pre- serving the structural and biochemical integrity of the components to which they are applied. This type of limitation, which is a familiar one in the biochemical field, is further complicated in the present case by the fact that the populations to be separated are not, like those dealt with usually by biochemists, composed of identical individual members. Most populations of subcellular particles are polydisperse with respect to all the physical properties which can be exploited for the purpose of fractionation and this heterogeneity is generally enhanced by the damage suffered by the particles during and after grinding of the tissue. This fact not only complicates separation, but also invalidates to some extent any non-quantitative preparative attempt as possibly leading to biased sampling of the population. These considerations make it clear that the resolving power of the techniques used in tissue fractionation is rarely commensurate with the complexity of their object and that great caution must be exercised in evaluating their outcome. Two types of techniques are available for this purpose, the morphological and the biochemical. Morphological methods are limited by the characteristics of the instrument used, by the difficulty of achieving perfect random sampling on a minute scale and by the lack, in many cases, of adequate criteria of identification, especially when structural details have become distorted by preparative procedures. Unless used with extreme care and thoroughness, they can serve only to identify the main component of a fraction and to provide a rough idea of the homogeneity of the preparation. But they cannot be trusted for the detection of rare particulate components nor of contaminants with dimensions below the resolving power of the instrument. For these reasons, when a preparation has been purified on the sole basis of morphological criteria and is then subjected to analytical measurements, it is necessary to consider carefully the probability of the measured property PLATE I. Electron-micrograph of a thin section of a lysosome-rich fraction isolated by isopycnic density gradient centrifugation in a glycogen-0.5 M sucrose gradient (Baudhuin & Beaufay, 1963). Most particles in the field are typical pericanalicular dense bodies (Rouiller, 1954; Novikoff, Beaufay & de Duve. 19.56). ( ,: 16,500)

PLATE II. Electron-micrograph of a thin section of a purified fraction concentrating mate oxidase, catalase and D-amino acid oxidase, isolated by isopycnic density gradient centrifugation in an aqueous sucrose gradient (Baudhuin & Beaufay, 1963). Most particles in the field correspond to the “microbodies” described by Gansler & Rouiller (1956) and Rouiller & Bernhard (1956). (X 16,500) PRINCIPLES OF TISSUE FRACTIONATION 41 truly belonging to the component forming the object of the purification. Let us take, for instance, a preparation of nuclei or mitochondria, which has passed the usual morphological tests of purity. Obviously, if light-scattering, nitrogen or total lipid content are measured on such a preparation there can be no doubt that the results obtained really apply to the nuclei or the mitochondria, since the possible contribution of contaminants to these bulk properties is necessarily a negligible one. Again, if one is dealing with a functional property which has clearly been ascribed to the major component by a variety of independent observations-such as, for instance, oxidative phosphorylation in a mitochondrial suspension-one may treat the results with some confidence, although in this case one already has to keep in mind the possibility of interference by contaminants. On the other hand, if the assay applies to a trace metal or to an as yet unlocalized enzymatic activity, the possibility that the constituent belongs to a minor component or is attached to the major one by an adsorption artifact deserves serious consideration. This point was brought home a number of years ago when it was found that many of the enzymatic activities found in allegedly pure nuclei are actually of cytoplasmic origin. It is also clearly illustrated by the observations which have been made in recent years on mitochondrial fractions from rat liver. In Table 1 are listed a number of enzymes all of which were found in our laboratory to be associated with the mitochondrial fraction to the extent of 65 or more per cent of their total tissue content. If interpreted on the basis of the morphological properties of the fraction, these results might easily have led to the conclusion that these enzymes are all localized largely in the mitochondria. In point of fact, this is true for only 7 out of the 18 enzymes listed. As will be indicated in more detail below, the others are associated with two distinct types of minor components of the preparation: the acid hydrolases with the lysosomes (for reviews, see de Duve, 1959; Novikoff, 1961b) or pericanalicular dense bodies (Plate I), urate oxidase, catalase and D-amino acid oxidase with another group of particles (de Duve et al., 1960; Baudhuin & Beaufay, 1963) described by morphologists under the name “microbodies” (Plate II). Although very hard to detect in the original mitochondrial preparation, both particles have been separated in relatively pure form by subfractionation of the latter. And they may not even be the only contaminants present, since there is evidence that mitochondrial fractions always include some microsomal elements and may also contain pinocytosis vacuoles (phagosomes, Straus, 1958). This example should suffice to demonstrate the hazards involved in relying exclusively on morphological criteria of purity in tissue fractionation work. Many others could already be given such as, for instance, the observations made by Whittaker (1959) and Gray & Whittaker (1960) on particulate 42 CHRISTIAN DE DUVE TABLE 1 Some enzymes occurring predominantly? in the mitochondrial fraction from rat liver

Host-particles Enzyme Reference:

j%Hydroxybutyrate dehydrogenase (2) Malate dehydrogenase (NAD dep.) (2) Succinate dehydrogenase (cyt. c) (1) Mitochondria Glutamate dehydrogenase (2) Cytochrome oxidase (1) Thiosulphate sulphurtransferase (rhodanese) (1) Alkaline deoxyribonuclease (2) Acid ribonuclease (1) Acid phosphatase (1) Acid deoxyribonuclease (1) /I-Galactosidase (3) Lysosomes a-Mannosidase (3) /%Acteylaminodeoxyglucosidase i3j j-Glucuronidase (1) Cathepsin (1) D-Ammo acid oxidase 14) Microbodies Urate oxidase (1) Catalase (4)

t Occurring to the extent of 65 or more per cent of their total tissue content in the sum of the heavy and light mitochondrial fractions isolated according to de Duve et al. (1955). $ References: (1) de Duve et al. (1955); (2) Beaufay, Bendall, Baudhuin & de Duve (1959a); (3) Sellinger, Beaufay, Jacques, Doyen & de Duve (1960); (4) Unpublished results reported briefly by de Duve (1960). fractions from guinea-pig brain, and one need only glance through a few randomly selected electron micrographs of tissue sections to realize how many structural components of as yet unknown biochemical significance are likely to be present in subcellular fractions. Biochemical measurements do not suffer from the limitations of morphological examinations and can usually be worked out to the required degree of specificity, sensitivity and accuracy. However, for a number of years there seemed to be no clear way of interpreting them in terms of structural components except by relating them to the available morphological data. Most preparative experiments have been conducted along this principle and even many of the results obtained in quantitative fractionation have been interpreted in a similar fashion, by simply identifying each fraction with its main morphological component. This is essentially what is done when a given enzyme or other biochemical constituent is reported as belonging to the nuclei to the extent of x per cent, to the mitochondria to the extent of y per PRINCIPLES OF TISSUE FRACTIONATION 43 cent, and so on. As discussed above, this process of identification is hazardous enough when applied to purified preparations; it is even less commendable when purification is further restricted by the requirement for quantitative recovery. Classical scheme Modified scheme

Cytochrome oxidose

Acid phosphatcse

Glucose-6- phosphotose

0 20 40 60 80 100 0 20 40 60 80 100 ‘b enzyme Ordinate =Relative speclflc activity = __ 46 nitrogen Absclsso= Nttrogen content,% of total mtroben FIG. 1. Distribution patterns of marker enzymes. Fractionation of 0.25 M sucrose homogenates from rat liver, according to Schneider (1948) (classical scheme) or to de Duve et al. (1955) (modified scheme). The fractions are represented along the abscissa in the order in which they are isolated. Classical scheme: nuclear (N), mitochondrial (M), rnicro- somal (P), supematant (S). Modified scheme: nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P), supematant (S). The finding that some enzymes belong exclusively to a given structural component, even though they may occur in several fractions, prompted a new approach in which the biochemical results acted as primary guides in the interpretation, independently of any morphological evidence (de Duve & Berthet, 1954; Appelmans et al., 1955; de Duve et al., 1955). To illustrate this approach, some of our early results are shown graphically in Fig. 1. On the left hand side of the Figure are depicted the distribution patterns of 44 CHRISTIAN DE DUVE cytochrome oxidase, acid phosphatase and glucose-6-phosphatase, as they would be found in a conventional fractionation of rat liver, according to Schneider (1948). Instead of taking these results as characterizing the enzymatic activities of the major component of each fraction, i.e. nuclei, mitochondria, microsomes and cell sap, they were interpreted as indicating the distribution amongst the fractions of the specific organelles bearing each enzyme. For instance, the distribution of cytochrome oxidase was taken to be that of the mitochondria, in conformity with the work of Hogeboom, Schneider & Striebich (1952), that of glucose-6-phosphataset to reflect that of microsomal elements, in agreement with the data of Hers, Berthet, Berthet & de Duve (1951). The fact that these enzymes were not concentrated exclusively in a single fraction was accepted as evidence of the inherent imper- fections of the separation methods used, an assumption which could be easily substantiated on theoretical grounds. To interpret the distribution of acid phosphatase, which turned out to be intermediate between that of the two marker enzymes, one had the choice between either accepting without further proof that this enzyme is located partly in the mitochondria and partly in the microsomes or, assuming that it too is associated with a single group of particles which, in the present case, could be neither mitochondria nor microsomes but did (by virtue of their sedimentation properties), sediment partly with the mitochondrial and partly with the microsomal fraction. The latter assumption had the advantage that it could be tested by experiments and these led to the finding that the difference in distribution between acid phosphatase and the other two enzymes could be enhanced considerably by modifying the fractionation scheme so as to include a small intermediate fraction containing the lighter mitochondria as well as the heavier microsomes. As shown in the right-hand half of Fig. 1, this fraction turned out to be particularly rich in acid phosphatase and to contain much more of this enzyme than could be expected from its content in mitochondria and microsomes, had acid phosphatase been associated homogeneously with these particles. These results were interpreted as supporting the working hypothesis and the latter was put onto increasingly firm ground as more and more enzymes were found to show distributions similar to that of acid phosphatase. The nature of the reasoning involved in this kind of approach becomes clearer when one considers the results obtained more recently by means of more refined techniques. By applying density gradient centrifugation to mitochondrial fractions from rat liver, Beaufay, Bendall, Baudhuin, Wattiaux

t Workers interested in a microsomal marker should remember that glucose&phosphatase is absent in many tissues. So far, it has been identified with certainty only in liver and kidney, where it is probably present in many species. It also occurs in the intestinal mucosa of guinea-pigs, but not of rats (Ginsburg & Hers, 1960). PRINCIPLES OF TISSUE FRACTIONATION 45 & de Duve (1959b) were able to construct the frequency distribution curves of sedimentation coefficients for a number of particulate enzymes. Their results brought to light, in a much more clearcut fashion than did the distribution patterns of Fig. 1, the existence of at least two distinct populations, one including most of the nitrogen of the preparation, cytochrome oxidase as well as several other enzymes and represented manifestly by the true mitochondria, the other comprising acid phosphatase and a number of other acid hydrolases, together with urate oxidase. To the latter group could also be added catalase, from the work of Thomson & Klipfel (1957) and, presumably D-amino acid oxidase, in view of the distribution experiments made by Paigen (1954) and in our own laboratory. These results provided strong corroborative evidence in favor of the existence of a second population of particles in mitochondrial fractions and, for a while, it was believed that all the enzymes showing the second type of distribution pattern were located in the same particles. This view had to be abandoned when further experiments performed by isopycnic centrifugation in sucrose and glycogen gradients showed that the second group of enzymes could itself be resolved into two quite distinct populations, one containing only the acid hydrolases and represented by the lysosomes, the other including the hydrogen-peroxide linked enzymes urate oxidase, catalase and D-amino acid oxidase (Beaufay et al., 1959b; de Duve et al., 1960). These investigations finally served to guide appropriate preparative attempts which led to the isolation of two relatively purified fractions suitable for morphological identification. In confirmation of previous biochemical and cytochemical investigations, the lysosomes were found to correspond to the pericanalicular dense bodies (Plate I), while the particles containing urate oxidase, catalase and D-amino acid oxidase were identified tentatively with the microbodies (Plate II). Considered a posteriori, the approach followed in these experiments is simply the standard way of the chemist. A mixture is first subjected to analyti- caI fractionation, preferabIy by several different methods, with no preconceived idea as to its contents. If the analyses disclose the presence of new, unidentified components, the information is exploited for preparative purposes, the unknown compounds are isolated and identified structurally. This is how, for instance, odd bases were discovered in nucleic acid digests. The only difference, in the present case, is that the method had to be freed to some extent from the context of a habit in which the analytical and preparative aspects of fractionation had not been clearly distinguished, and also that it dealt, not with well-defined molecular species, but with complex particles which could be identified only by one or a few of their constituents. The latter fact subordinated the approach to two working hypotheses, which have been 46 CHRISTIAN DE DUVE called the postulate of single location and that of enzymic homogeneity (de Duve, 1957, 1960). The first postulate, which assumes that “a given enzyme belongs to a single intracellular component in the living cell” (de Duve & Berthet, 1954), although of great practical value in many cases, is not really essential, as the method can also be applied successfully to bimodal or multimodal distributions. The second one, however, is of primary importance. As first defined, it presupposes that “granules of a given population are enzymically homogeneous, or at least cannot beseparated by centrifuging into subgroups diflering significantly in relative enzymic content” (de Duve et al., 1955). Obviously, this condition has to be met if we want to use the enzymes as valid indicators of their host particles. On the whole, these postulates have so far stood the test of time relatively well. They are not verified in a perfect manner in all cases, but the discrepan- cies which have been observed until now have not been sufficiently serious to invalidate the approach. In any case, as discussed elsewhere (de Duve, 1957), the postulates do not have to be valid in all cases to be useful, since the experimental approaches which they inspire are such as to prove them eventually wrong if they are, meanwhile providing valuable additional information. In conclusion, preparative fractionation, when relying solely on morphological examination for purity control, is adequate only for the determination of bulk properties of intracellular components. It may lead to serious errors when used for the analysis of their enzymatic equipment or of their content in other trace constituents. The pseudo-preparative analytical approach, exemplified by the usual method of quantitative fractionation, should be dissociated largely from its misleading preparative aspect and considered principally as an analytical tool of low resolving power, only suitable in most cases for a preliminary attack of a problem. The purely analytical approach, especially when conducted with modern methods and with the help of marker enzymes, is the only one to be unrestricted in its scope and should be given precedence over the others. As information accumulates, it may eventually lead to the development of new preparative techniques, as in the investigations described here, or in the studies of Gray & Whittaker (1960) which have led to the isolation of nerve endings from guinea-pig brain. Or it may provide more solid criteria of purity to verify the efficiency of existing preparative methods, as, for instance, in the work of Chauveau, MOUE & Rouiller (1957) who applied a number of biochemical controls to evaluate their procedure for the isolation of nuclei.

Choice of Methods Although successful use occasionally has been made of chromatography, electrophoresis, or phase partition in the fractionation of subcellular com- PRINCIPLES OF TISSUE FRACTIONATION 47 ponents, the methods most widely employed so far have been centrifugal, depending on differences in either sedimentation rate or density between the populations to be separated. In devising appropriate techniques of this kind, it is important to remember that the physical characteristics of most subcellular particles are not invariable but depend in a critical fashion on the composition of the suspending medium.

I I I I I I I I I I I I I w

\,+Lysosomes \ \ _I

I I I I I I I 0.02 0.04 0.06 0,02 0.04 0.06 Mole fraction of sucrose FIG. 2. Influence of sucrose concentration and of solvent on density of cytoplasmic particles. Constructed from the data of Beaufay& Berthet (1963).The curvesfor mito- chondriashow a discontinuityat a high sucroseconcentration, reflecting the transition to the so-called“dense state” of theseparticles.

Moreover, the relationship between medium composition and particle properties is not necessarily the same for each group of particles, so that particles which are not separable in a given medium may become so in another. This point is illustrated by the curves of Fig. 2, which show the manner in which the sucrose concentration of the medium and the nature of the solvent water affect the difference in density between cytoplasmic particles and medium. These curves were constructed according to a theoretical equation worked out by de Duve et al. (1959), and with the experimental values established by Beaufay & Berthet (1963). The latter publication should be consulted for details. 48 CHRISTIAN DE DUVE As shown by Fig. 2, the degree of separability of the three groups of particles studied varies with the composition of the medium, and the data obtained make it possible to select the system best suited for a given type of separation. Obviously, medium composition can be altered in many other ways, so that even within the narrow limits set by the requirements for structural and biochemical integrity of fragile subcellular particles, the investigator still remains free to modify a number of variables in his search for a medium adapted to his particular problem. In this respect, centrifugal fractionation has potentialities comparable to those of other fractionation techniques of high resolution, such as electrophoresis or chromatography, but which have not yet been exploited as they deserve to be. The analogy with other fractionation methods is also apparent in another conclusion which arises from the above considerations, namely that inseparability of two components should not be declared too hastily on the basis of a single type of experiment. Only when the association is found to persist in several different media does it become legitimate to suspect that it has a true structural basis. Experimental Design and Interpretation Any tissue fractionation experiment proceeds in three successive steps: homogenization, fractionation and analysis. These have to be retraced backwards by the investigator when he attempts to interpret his analytical data in terms of properties of the original intact cells or tissue. A rigorous discipline must be observed in this process of mental reconstruction. In particular, it is essential that each step be clearly dissociated from the others without any overhasty extrapolation, since the criteria of judgment are of a different nature at each stage. Many errors of interpretation on record in the literature can be related to a failure to comply with this rule. The first stage of the interpretation must be restricted to the assignment of quantitative biochemical properties to the fractions, irrespectively of their accepted or suspected cytological significance. If morphological examinations have also been performed, their results must be expressed separately, in qualitative or semi-quantitative terms, but no attempt to correlate the two sets of data should be made at this stage. The criteria involved here are mainly biochemical and concern the accuracy and specificity of the assays and the manner in which they are affected by structural factors or by the presence or absence of specific substances. As a rule, a fair amount of additional work is required to provide the necessary data. For instance, when enzymes are measured, kinetic experiments must be carried out in order to establish optimum conditions and to verify the essential requirement for proportionality between reaction velocity and enzyme concentration. Strictly speaking, although this is rarely done, such measure- PRINCIPLES OF TISSUE FRACTIONATION 49 ments should be performed on all the fractions and on mixtures of them in order to ascertain the possible existence of interactions due, for instance, to the unequal distribution of cofactors or of inhibitors. In some cases more elaborate enzymological investigations designed to test the presence of more than one enzymic species may be indicated, especially when the observed distribution appears to be a complex one. Such experiments are actuated by the postulate of single location and have already provided valuable information supporting this postulate in a number of cases. For instance, bimodal distributions have been broken down to the unimodal distributions of two distinct homologous enzymes for malate dehydrogenase in ox heart (Wieland, Pfleiderer, Haupt & Worrier, 1959-60; Siegel & Englard, 1960), 17 /3-hydroxysteroid dehydrogenase in guinea-pig liver (Endahl, Kochakian & Hamm, 1960; Villee & Spencer, 1960), aspartate aminotransferase in rat liver (Rosenthal, Thind & Conger, 1960; Eichel & Bukovsky, 1961) deoxyribonuclease in rat liver (Beaufay et al., 1959b), arylsulphatase in the liver of several species (Roy, 1958), glutaminase in rat liver (Shepherd & Kalnitsky, 1951), aconitate hydratase in rat liver (Dickman & Speyer, 1954), and several reductase, diaphorase and phosphatase activities (for a review, see de Duve, Wattiaux & Baudhuin, 1962). Another test of considerable importance concerns the existence of structure- linked latency for particulate enzymes. This has now been found to be a widely distributed phenomenon, explainable in most well-documented cases by the presence of a barrier, generally the membrane of the particle, restricting the accessibility of enzymes located within a particle to external substrates. In quantitative measurements of the total activity of a fraction, this barrier must be removed completely and in a manner which will neither inhibit nor inactivate the enzyme. Special experiments are required to set up appropriate conditions fulfilling this aim. Such experiments may also be of great help in the further interpretation of the results, for the sensitivity to agents which cause the release of bound enzymes varies significantly from one type of particle to another. For example, it has been observed by Bendall & de Duve (1960) that mitochondria are more resistant than lysosomes to repeated freezing and thawing and exposure to hypotonic media. In recent unpublished experiments, Baudhuin has found that they are also considerably less sus- ceptible than lysosomes to disruption by digitonin. Microbodies, as investi- gated by their ability to release catalase, occupy an intermediate position in this respect and also differ from the other two groups of particles by their almost complete resistance to osmotic disruption. Finally, it is important at this stage of the reconstruction process to establish a “balance-sheet” allowing to estimate the losses (or gains) sustained in the course of fractionation and to express the biochemical T.B. 4 50 CHRISTIAN DE DUVE results obtained on the fractions in terms of the values found for the unfractionated starting material. The importance of this requirement has been strongly emphasized by early workers (see Schneider & Hogeboom, 1951; Hogeboom, Schneider & Striebich, 1953) and subsequent findings have generally confirmed the validity of their contention. Most investigators have subscribed to this point of view as applied to quantitative fractionation experiments and there are very few publications in which recovery values are not reported for experiments of this type. However, the significance of the balance-sheet has been questioned in relation to preparative experiments designed for the purification of a given intracellular component. For instance, it has been pointed out (see Allfrey, 1959) that the presence in a purified nuclear preparation of less than 10% of the total activity of an enzyme does not necessarily mean that the enzyme is not a true nuclear component. While this is of course correct, the fact remains that the chances of cytoplasmic contamination being involved increase as the percentage of activity observed decreases. The information provided by the balance-sheet is therefore of considerable usefulness in guiding the subsequent interpretation of the results in cytological terms. To disregard it in preparative fractionation is particularly unwarranted, in view of the relative inadequacy of morphological criteria for purity control. In the second stage of interpretation, the fractions and their biochemical properties are related to the components of the homogenate. Even here, it is advisable to refrain from any premature cytological identification of these components and to consider them exclusively in terms of their physical characteristics, as revealed by their behaviour in the centrifuge. This is what is done, ideally, in density gradient centrifugation, when frequency distribution curves are constructed as a function of one of the variables, sedimentation coefficient or density, on which the fractionation procedure is based. Dia- grams of the type depicted in Fig. 1 can be looked at in a similar fashion. Since the fractions are aligned along the abscissa in the order of decreasing sedimentation coefficient, the distribution patterns may be considered as very rough and distorted estimates of the frequency distribution curves of sedimentation coefficients. In some publications, the results are related to particle size rather than to sedimentation coefficient. While serving a similar purpose, this practice may be somewhat misleading, since the derivation of particle size requires a knowledge of the shape and density of the particle, which are rarely known with any accuracy. The construction of distribution curves or diagrams requires a thorough understanding of the physical principles underlying sedimentation in a centrifugal field and a careful control of the variables such as time, field, temperature, medium composition, etc., which determine the behaviour of particles in the PRINCIPLES OF TISSUE FRACTIONATION 51 experiment as it has been carried out. Except for that, it still remains a purely factual description of the results and involves no preconceived idea as to the nature or properties of the material which has been fractionated. To go a step further, it is necessary to make some assumption concerning the relationship between the assayed biochemical constituents and their supporting particles. This is where it becomes useful, in agreement with the analytical point of view developed above, to introduce the postulate of biochemical homogeneity. According to this postulate, the distribution of a biochemical constituent, usually an enzyme, is taken to reflect the mass distribution of the particles with which it is associated. Therefore, once it is accepted as a working hypothesis, the distribution curves can now be interpreted tentatively as being representative of the actual particles, expressed in terms of mass or of some more or less equivalent unit such as total amount of nitrogen or protein. From the shape of the curves and from their position on the diagram, one can now derive a great deal of information concerning the physical properties of the individual particles and their statistical distribution within the population. In addition, when several constituents or enzymes have been measured simultaneously, comparison of their distribution curves will indicate whether they belong to different particles or whether they could be associated together with the same particles. As pointed out above, association in a single type of experiment does not necessarily prove true structural association. In the last step of the interpretation, which is the most difficult one, the attempt must be made to express the results in cytological terms and to identify as specific intracellular organelles or structures the as yet anonymous particles characterized in the preceding step. This requires a good knowledge of the morphology of the cells or tissue under investigation as well as an understanding of the mechanical, physical and chemical events which are likely to take place in the course of a fractionation experiment, especially during the initial highly traumatic process of homogenization. It is useful, in this last stage of the interpretation, to attempt to dispose of any apparent anomaly in the observed distribution diagrams. Such anomalies include bimodal or multimodal particulate distributions and the presence of the same constituent in both the soluble and in particulate fractions. The postulate of single location provides a valuable guide for these attempts. As already pointed out above, complex enzyme distributions may reflect the existence of more than one enzyme species exhibiting activity under the conditions of the assays and this may be tested by appropriate enzymological experiments. When a specific constituent occurs in soluble and in particulate form its location, if truly single, may be either the cell sap, in which case adsorption is responsible for its association with particles, or a fragile particle from which it is easily released in the course of homogenization or fractiona- 4-z 52 CHRISTIAN DE DUVE tion. Elution experiments, especially with different ionic media, may serve to test for an adsorption artifact (for an example, see the investigations of Rosenthal, Gottlieb, Gorry & Vars (1956) on the elution of thiosulphate sulphurtransferase (rhodanese) from nuclear fractions), while the existence of structure-linked latency may help to establish the significance of the bind- ing of an enzyme to particles. An important point to keep in mind when checking for such artifacts is that many of them can be ascribed to events which take place during the initial homogenization of the tissue. Several techniques are now available for accomplishing this operation and considerable gains in accuracy and resolution can sometimes be obtained by comparative experiments designed to set up optimum grinding conditions for the particular material under investigation. Logically, such tests should constitute the first step in fractionation work and many authors have in fact devoted a great deal of time and effort to this aspect of the problem, usually with morphological control as guiding criterion. Here again, the value of biochemical tests should not be forgotten. In particular, the degree of latency of enzymes associated with fragile particles such as, for instance, the lysosomal hydrolases, may provide an easy evaluation of mechanical damage. An example of such controls is given by the experiments of Greenbaum, Slater & Wang (1960) on mammary gland. Since biochemical knowledge often comes as a result of fractionation experiments, a reinvestigation of homogenization conditions at a later stage may often be indicated. When the experimental picture has been corrected as much as possible for the distortions introduced by artifacts, it is now ready for reinterpretation in terms of cytological components. Several distinct pieces of information can be brought together to achieve this aim. The most direct is provided by the results of any morphological examinations performed on the fractions and by the con- frontation of the physical characteristics deduced from the behaviour of the particles in the centrifuge with the known structural properties of the components which can be seen in the intact cells or tissues. In addition, as shown in the examples discussed in this paper, the use of enzymes or of other specific components of known location as markers for their host-particles can also be extremely valuable. So are the data on structure-linked latency, especially when they include details on the mode of release of enzymes under the influence of different unmasking agents. It is important, throughout this process of interpretation, to remember that the initial picture derived from the biochemical results rests on the postulate of biochemical homogeneity and is therefore falsified to the extent that the particles under consideration are heterogeneous with respect to the assayed constituents. It is also necessary at some stage of this interpreta- PRINCIPLES OF TISSUE FRACTIONATION 53 tion to take into account the cellular heterogeneity of the fractionated material. The latter problem is one of the more serious ones facing the investigator working with solid tissues, and it is likely to become more so as greater resolution is obtained. Some attempts have been made to solve it by first converting the tissue to a suspension of intact cells and separating the latter by a preliminary fractionation (see for instance Wattiaux, Baudhuin, Berleur & de Duve, 1956). However, the techniques presently available for this purpose are not too satisfactory. In some exceptional cases, it has been possible to eliminate more or less selectively one type of cell, as in the numerous investigations on the lymphoid tissues from X-irradiated animals (for a review see Eichel & Roth, 1960); but this method is complicated by the possible effects exerted by the eradicating treatment on the remaining cells. Probably, this is one of the areas where close collaboration with the visual cytochemist will be most fruitful, since staining techniques of even relatively poor resolution may suffice for the localization of constituents at the cellular level. It will be clear from the above considerations that tissue fractionation does not lend itself readily to casual or occasional applications nor to rigid coding for routine use. Although common to many types of biochemical fractionation, this challenging character of the method has not yet been generally recognized. For instance, it has become customary in investigations dealing with the purification and characterization of enzymes to include data on their intracellular distribution. This is a highly commendable practice, which, unfortunately, is rarely pursued with the necessary perseverance and imagina- tion. In many cases, the scheme developed by Schneider (1948) for rat liver is followed more or less blindly, irrespectively of the nature of the starting material, and the results are given simply in the form of a table listing the distribution of the enzyme between “nuclei”, “mitochondria”, “microsomes” and “supernatant”, with little or no critical appraisal of their significance. If the enzyme is recovered in overwhelming amounts in the supernatant fraction, there is obviously little more that can be done. But if particulate components appear to be involved, such experiments are generally of little value, unless they lead to further investigations. In these, as in the fractionation of biochemical constituents, it is often necessary to use a multi- plicity of approaches and, especially, to follow the self-correcting technique so familiar, for instance, to the protein chemist, whereby the information gained is continuously fed back to modify the experimental design until an un- equivocal result has been obtained. Each individual problem may have different requirements in this respect and luck or inspiration may often turn out to be more helpful than adherence to a complicated set of rules. However, logic has to be respected. In the author’s opinion, this is done most easily by always keeping clear the essential distinction between the subcellular 54 CHRISTIAN DE DUVE fractions as they are isolated and analysed, the populations of particulate aggregates as they are present in the homogenate and react to the fractionation procedure, and the intracellular organelles or structures as they occur within the cells. One can often avoid an erroneous interpretation or escape being side-tracked into a blind alley by taking care not to jump unnecessarily or prematurely from one to the other.

Nomenclature The logical requirements stressed in the preceding section can only be satisfied with the help of a precise and unambiguous vocabulary. Unfortu- nately, the language of cytology is sadly lacking in this respect. After having long been hampered by the paucity and poorness of the criteria on which to base its definitions, it has now reached an even greater state of chaos in its efforts to keep pace with the rapid developments that have taken place in such diverse disciplines as electron microscopy, visual cytochemistry and subcellular biochemistry. At the present time, it may be said to include very few terms carrying exactly the same connotation in all interested areas and one even wonders, when perusing the current literature, to what extent the logical inconsistencies which have been denounced here are the result rather than the cause of this semantic confusion. If we take, for instance, a word such as “mitochondria”, we find that its morphological definition has followed a long and hesitant course until the findings of electron microscopists have finally provided it with a relatively clear set of criteria (see Novikoff, 1961a). However, this is a very recent happening and only a few years ago many morphologists were still ready to extend the name rather indiscriminately to a large number of different cytoplasmic bodies (see for instance Rouiller, 1960). In the meantime, biochemistry has made its fundamental contributions to our knowledge of mitochondria by showing first that these particles can be separated from tissue homogenates by means of differential centrifugation and demonstrating further that they are the main site of oxidative processes and of the associated phosphorylations. With the development of specific staining reactions for oxidative enzymes, visual cytochemistry has provided a valuable and essential link between the morphological and biochemical observations, thus completing the experimental basis for an accurate definition of mitochondria as specific cytoplasmic organelles with typical structural and functional properties. From now on, there should be little difficulty in recognizing mitochondria wherever they are present and we should only expect our knowledge of these bodies to increase in scope and detail as more evidence accumulates. PRINCIPLES OF TISSUE FRACTIONATION 55 Unfortunately, it has become customary amongst biochemists to designate as “mitochondria” the tissue fractions containing these organelles as major component, or even, in some cases, any cell fraction isolated under the conditions set out for the separation of rat liver mitochondria. This practice, which has been sanctioned by the editors of most periodicals, brings in an unnecessary and greatly misleading ambiguity into any discussion of the properties of such fractions. If adopted in the present paper, for instance, it would have led to the nonsensical question as to which mitochondria are not mitochondria. This kind of confusion is likely to creep in, not only in the writings, but sometimes also in the reasoning of an investigator whenever he substitutes without adequate justification the name of a well-defined intracellular organelle or body for that of the fraction in which it is known, believed or surmised to be concentrated in relatively pure form. Quite obviously, there are cases where the substitution is perfectly justified, for instance, in light- scattering work on mitochondrial suspensions. There are undoubtedly many others where, though not justified, it will turn out to be correct, since on a purely probabilistic basis the properties of a fraction have a greater chance of belonging to the major than to a minor component of the preparation. However, there are also a number of instances where the substitution will be wrong and will practically bar the way to a correct interpretation of the results. In other words, the choice between a specific cytological term such as “nuclei” or “mitochondria” and an operational one such as “nuclear fraction”, “mitochondrial fraction” or “cytoplasmic particles” actually involves an essential judgment of values on the part of the investigator and should not be made without due consideration. The rule to be followed is a very simple one. Even though they may be more cumbersome, use the non- commital periphrases whenever there can be any doubt with respect to the true cytological identity of the bodies referred to. The discovery of new intracellular entities either by morphologists or by biochemists has introduced even greater difficulties in the current cytological nomenclature. Lysosomes are a case at hand. As reported elsewhere (de Duve, 1959), “the term has this peculiarity that it refers to what is believed to be a morphologically distinct entity, defined on the basis of purely biochemical data”. It designates a particle, first identified in rat liver, containing a number of soluble acid hydrolases and surrounded by a membrane which restricts the accessibility of the internal enzymes to external substrates. Lytic bodies answering this definition have now been detected in a great variety of tissue preparations and the name proposed has undoubtedly been of some help in focusing attention upon their basic biochemical and functional similarities. However, this usefulness may soon be offset by the liberal 56 CHRISTIAN DE DUVE manner in which the original definition is being interpreted by different authors. As in the case of mitochondria, but even less justifiably so, the term “lysosomes” has already been used by a number of biochemists to designate lysosome-rich fractions (still containing these particles as minor component, however) or even uncharacterized fractions which happen to have been isolated by the procedure which, when applied to rat liver, furnishes a lysosome-rich fraction. Unfortunately, the latter fraction was originally labelled L, for “light mitochondrial fraction” (de Duve et al., 1955). This has now been reinterpreted to lysosomes and the name has already degenerated to such an extent as to have been used in at least two instances to designate a fraction which the authors’ own analyses showed to be poor in acid hydrolases. Recent progress in the morphological identification of lysosomes has brought to light an unexpected complication in the terminology. It appears that, unlike other cytoplasmic entities, Iysosomes may not have uniform structural features in different cells nor even, possibly, in the same cell. This arises from the fact that the biochemical concept of a bagful of acid hydrolases probably covers a number of entities which, though functionally related, may be very different in appearance, ranging all the way from storage granules for the enzymes, through various digestive and autolytic vacuoles containing material in the process of digestion, down to residual bodies loaded with remnants of various nature. Eventually, these distinctions will have to form the basis of a more detailed nomenclature. In the meantime, the polymorphism of lysosomes has been responsible for some confusion since it has led a number of workers to apply the name to various cytoplasmic bodies of unknown nature without evidence that they actually contain acid hydrolases. Even the practice of designating as lysosomes any structure showing a positive reaction for acid phosphatase (Novikoff, 1961b), although much more justified, does remain open to some criticism since there is no evidence that acid phosphatase is always confined to particles of the lysosome type. Possibly the most troublesome word in our cytological vocabulary is represented by the term “microsomes”. Originally proposed as synonym of “small granules”, it had a purely operational significance and served simply as an inclusive denomination for all subcellular entities requiring a relatively high centrifugal force for complete sedimentation. With the demonstration that some enzymes are largely associated with the microsomal fraction and may be recovered almost entirely with this fraction when suitable precautions are taken to prevent losses by agglutination or cosedimentation with the larger particles, the term began to carry a more specific meaning and was PRINCIPLES OF TISSUE FRACTIONATION 57 used by many biochemists to designate what was believed to be a distinct group of intracellular components. This practice, which was adopted in the author’s laboratory in relation with the use of glucose-6-phosphatase as marker enzyme, was encouraged by the widespread application of the suffix “some” to specific bodies or structures; it led to a distinction between “microsomes” and “microsomal fraction”, the former being considered as the major component of the latter. This view now has to be revised in the light of recent electron-microscope studies, which have shown microsomal fractions to be made largely of membraneous elements derived from the endoplasmic reticulum and possibly from related structures such as the Golgi apparatus. These membranes are either of the smooth or of the rough type, the latter being distinguished by attached ribonucleoprotein particles. So far, most microsomal enzymes have been found to belong to the membranes, but indications have already been obtained that some of them may be exclusively associated with one or the other type of membrane. On the other hand, the ribonucleoprotein particles have been separated in relatively pure form, not only from microsomal fractions but also from cell types which contain little or no endoplasmic reticulum and in which they appear to occur freely in the cytoplasm. They have been shown to be the main site of protein synthesis and have been named “ribosomes”. As a result of these findings, the term “microsomes” has become extremely hard to define since it has lost part of its purely operational significance, while gaining nothing in cytological precision. There is little hope that it will disappear from our vocabulary, but it is hoped that it will be replaced more and more frequently by the expression “microsomal fraction”, which is less ambiguous. As our knowledge increases, it will then become possible to distinguish as components of this fraction the ribosomes and the various types of membraneous elements which will come to be identified morphologically and biochemically. In addition, it will be easier to refer to the minor components, such as the lysosomes, the phagosomes and the microbodies which, though always present in relatively large amounts in microsomal fractions, at least from rat liver, have never been included under the name “microsomes”. The examples discussed above will suffice to illustrate the semantic difficulties which beset workers engaged in modern cytological work as well as their readers. Perhaps the situation should not be overdramatized. Nor should one attempt too seriously to encase our present vocabulary into too rigid a framework of definitions. For the first time in the history of cytology, the organization of the cell is being explored in a somewhat coordinated manner by members of traditionally separated disciplines, It is inevitable 58 CHRISTIAN DE DUVE that from the intermixing of jargons some degree of confusion should arise. However, out of this process, a universal language of cell biology is being born and it is preferable to maintain it in a sufficiently fluid state until enough data become available for the codification of its vocabulary. In the meantime, the language must as least satisfy the requirements of logic and provide an adequate terminology for faultless reasoning. In the field of tissue fractionation, this is easily accomplished by always using different words to designate subcellular fractions and intracellular organelles or components.

The author is indebted to Drs J. Berthet and H. Beaufay for their valuable suggestionsin the preparation of this paper. The investigationsreferred to have been supported by grants from the Fonds National de la RechercheScientifique, the Centre Interuniversitaire de Recherchessur la CroissanceNormale et Patho- logique, the Centre Interuniversitaire de RecherchesEnzymologiques, the Rocke- feller Foundation, the Lilly ResearchLaboratories and the U.S. Public Health Service (grant No. RG-8705).

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