HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Liebert, Inc., Publishers

Monoclonal Antibodies to the Human Multicatalytic Proteinase (Proteasome) MAJ-BRITT

KALTOFT,13 CLAUS KOCH,2 WOLFGANG UERKVITZ,1

and

KLAVS B. HENDIL1

'August Krogh Institute, University of Copenhagen,

13 Universitetsparken, DK 2100 Denmark and 2The State Serum Institute, 80 Amager Boulevard, DK 2300, Copenhagen S Institut de Biotechnologie, Laboratoire de Génétique Microbienne, Domaine de Vilvert, F-78, Jouy-en Josas Cedex, France

Copenhagen O,

3INRA,

ABSTRACT

Multicatalytic proteinase is an intracellular enzyme composed of at least 12 different subunits. Seven murine hybridoma cell lines secreting antibodies to human multicatalytic proteinase (MCP) were established. The antibodies reacted with 4 different subunits of the oligomeric protein. Three of the antibodies shown bound to identical or closely spaced epitopes on the largest subunit, as by binding competition. Some of the antibodies cross-reacted with MCP from rat or rabbit, but none with lobster MCP. Glycoprotein components could not be rabbit detected in human MCP. The monoclonal antibodies and two polyclonal antibodies did not specifically inhibit the enzymatic activity of human MCI'. Electrophoretic analysis of MCP immunoprecipitated from human placenta, liver, kidney, or HeLa eel] extracts with antibodies to 3 different subunits suggested that the subunit compositions are very similar or identical. INTRODUCTION

F.ukaryotic cells contain a multicatalytic proteinase (MCP) with an Mr of about 740 kDa (1) In mammals the proteinase consists of about 12 different subunits with Mr in the range 21 four 32 KDa. The subunits are arranged in rings, that are stacked upon each other to form a hollow cylinder (2). MCP has 4 distinct proteolytic specificities (3) and cleave peptide bonds on the carboxyl The side of basic, acidic, and liydrophobic amino acids. (Reviewed in (4-5)). proteinase is the catalytic core in a larger protein complex that degrades proteins after their coupling to ubiqu.it in (6-9). The MCP is identical with prosomes that repress translation and may be endowed with other activities (5). We have produced monoclonal antibodies that are specific for certain individual subunits. These antibodies were used to immunoprecipitate MCP from various human cells and tissues. It has been suggested that MCP shows structural heterogeneity but our results imply that there are few, if any, variations in subunit composition of MCP among diverse human cell types. -

Abbreviations: MCP,

multicatalytic proteinase. 507

MATERIALS AND METHODS

Materials The following buffers were used: Buffer A: 137 mM NaCl, 2.7 mM KC1, 5 mM NaN3, 4.1 mM Na2HP04, 0.73 mM KH2P04, pH 7.4; Buffer B: 154 mM NaCl, 0.1 mM EDTA, 1.5 mM NaII2P04, 8.5 mM Na2HP04, 0.05 % Tween 20, pH 7.5; Buffer C: 48 mM Tris, 39 mM glycine, 0.01 X SDS, 20 %(v/v) methanol; Buffer D: 50 mM Tris/HCl, 150 mM NaCl, 5 mM NaN3, 0.01 X Tween 20, pH 7.4; Buffer E: 500 mM NaCl, 50 mM Tris/HCl, 5 mM NaN3, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, 0.05 X Tween 20, pH 7.4; Buffer F: 20 mM Tris/HCl, 20 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 20% (v/v) glycerol; Buffer G is buffer F with 1 M sucrose, 0.5 % Triton X-100 and 0.1 % SDS.

and their sources were: Biotin-conjugated lectins and rabbit (Kem-En-Tec, Copenhagen); peroxidase-conjugated to mouse immunoglobulin and peroxidase-conjugated streptavidin (Dako, Copenhagen); fluorogenic substrates (Cambridge Research Chemicals, Northwich, England); Ampholines (Pharmacia-LKB, Uppsala, Sweden). Other biochemicals were purchased from Sigma (St. Louis, MO, USA). HeLa cells were grown in Earle's MEM with 5 X newborn-calf serum (Glbco, Paisley, Scotland). MCP from lobster and rat were generously provided by Dr. Donald Mykles (Ft. Collins, CO, USA) and Dr. Burkhardt Dahlmann (Dusseldorf, FRG), respectively. The

protein antibody

materials

A-agarose

Purification and assay of_MCP MCP was purified essentially as described (10) but from human placenta. Alternatively MCP from human placenta or rabbit liver was purified on a column with immobilized antibody MCP21 (11). Enzyme activities were measured with or succinyl -ala-ala-phebenzyloxocarbonyl-ala- arg-firg-methy 1 coumarylam ide

methylcoumarylamide (11).

Production of monoclonal antibodies Female Balb/c mice were, immunized by intraperitoneal injection« of human emulsified in Freund's adjuvant. Complete adjuvant was used for the first incomplete Freund's adjuvant for the following Immunizations. After 4 injections with 2-week intervals, each time with 50 Mg MCP, the spleen cells were were fused with Sp2/0-Agl4 myeloma cells and the resulting hybridomrs cells screened by F.LTSA and cloned at lerist twice by limiting dilution, all according to standard methods (12). Antibodies were purified from peritoneal fluid from hybridoma beating mice on protein A agarose (12). MCP and

-

ELISA Microtiter plates (Nunc, Roskilde, Denmark) were incubated for at least 24 h with MCP diluted to 3 ug/ml buffer A (100 ul/well) followed by a 30 min incubation with 5 X fatfree drymilk in buffer A. The plates were washed 3 times in buffer B, incubated overnight with antibodies diluted in buffer B with 1 X calf serum (100 ul/well), washed 3 times in buffer B and incubated for 1 h with 100 ul/wel] of peroxidase-labelled rabbit anlibody to mouse immunoglobulins (1:1000 in buffer B with 1 X calf serum). After 3 washes in buffer B, bound peroxidase was asssyed with o-phenylenediamine/ Il202. Colour development was read at 490 nm in a microplate photometer. Antibody isotype was determined by ELISA on microtiter plates with adsorbed MCP using isotype specific antisera from Biorad (Richmond, CA, USA).

Binding competitIon assay of antibodies for binding to MCP was determined by ELISA (13): in microtiter plates with adsorbed MCP were incubated with saturating amounts of monoclonal antibodies either alone or in combination with saturating amounts of each of the other antibodies. The amount of monoclonal antibody bound to the adsorbed MCP was then measured with peroxidase-label led rabbit antibody

Competition

wells

508

to

mouse

antibody

immunoglobulin. The. binding of monoclonal antibodies antibody B was expressed as

from

a

mixture

of

A and

(A+B) * 2 (A) + (B) where (A + B) is the absorbance obtained with a mixture of the two antibodies, and (A) and (B) are the absorbances measured for binding of each of the monoclonal antibodies alone. A value of 2 is expected with antibodies that do not compete, and a value of 1 results if the antibodies show strong competition.

Electrophoresis and blotting SDS-PAGE was carried out in slab gels with 12^ % (w/v) acrylamide. For dimensional PAGE (14) MCP (20 ug/gel) was denatured in 8 M urea and separated by isoelectric focusing in a 50:50 mixture of Pharma.lyte 3-10 and Ampholine 5-7. Separation in the second dimension was attained by SDS-PAGE in a 12i % acrylamide gel (7x8 cm). The proteins were blotted onto nitrocellulose filters (BA83, Schleicher & Schuell, Dassel, Germany) by electrophoresis in two

buffer C in a semi-dry system. The blots were stained reversibly in Ponceau S and the protein spots marked on transparent paper. The filters were blocked either with 5 % fatfree drymilk in buffer A (before incubation with antibodies) or by incubation in 2 X Tween 20 for 2 mln (for detection of glycoproteins

(15)).

Detection of

an

tigen s

The

nitrocellulose filters were incubated overnight at room temperature with hybridoma culture supernatants diluted in buffer D (1:100 or 1:300). The filters were washed 3 times in buffer D and bound antibody was detected by incubation with peroxidase-coupled rabbit antibody to mouse immunoglobulins (1:1000 in buffer D) followed by incubation with H202 and tetramethylbenzidine as a chromogenic substrate.

Glycoprotein detection Blots

incubated for 18 h at room temperature with 0.25 pg bi.otlnper ml of buffer E. The filters were then washed 3 times in the same buffer. Lectins were detected with peroxidase-label1ed streptavidin (1: 10000 in buffer E (15) followed by incubation with H202/tet.ramet hylbenzidine. In an alternative attempt to detect glycoproteins, blots were incubated with NaI04 followed by peroxidase hydrazide as described (16), and then with were

coupled lectins

H202/tetramel:hylbenzid!ne. Immunoprecipit at ion

Monoclonal antibodies were, coupled to CNBr-nctivated Sepharose CL 4B to 2.0 mg/ml. Cells and tissues were homogenized in 3 volumes of 250 mM 5 mM EDTA, 0.5 mM phenylmet.hy 1 su 1 fony 1 fluoride, pH 7.4. HeLa cells sucrose, were disrupted in a Dounce homogenizer. Human liver and kidney, obtained by autopsy, and fresh placenta, were homogenized in a Potter-Elvehjem homogenizer. The homogenntes were spun for 30 min in a MSE 10X10 rotor (gav~ 116 000 g) and the supernatants diluted with one volume of buffer F. Aliquots of 2.5 ml of the diluted supernatants were tumbled for 3 h at 4°C with 0.5 ml of Sepharose with immobilized monoclonal antibodies. The Sepharose beads were layered on top of 10 ml of buffer G and centrifuged (200 xg, 5 min) reliefs were resuspended in 1 ml of buffer F and centrifuged through another 10 ml of buffer G as before, The centrifugat.ion was repeated and the beads were suspended into 10 ml of buffer A and centrifuged again. After aspiration of supernatant, antigen was eluted from the pellet with 2 ml of 0.2 X SDS at 100°C for 2 min. The beads were centrifuged again and re-extracted with 2 ml of water at I00°C for 5 min. The two eluates were combined, lyophilized and redissolved in 150 ul of SDS sample buffer. 15 ul were electrophoresed and the gels were silver stained. Samples of 5 1.5

-

-

509

RESULTS MCP purified from human placenta could be resolved by 2 dimensional electrophoresis into at least 12 subunits. The subunits have been numbered as shown in Fig. 1. Some preparations of MCP also contained a protein with the same isoelectric point as subunit 12, but with slightly lower Mr. The poorly focussed proteins, 3 and 3', varied inversely in staining intensity in 6 separate preparations of MCP, and they have identical Mr but different pi values. These spots may therefore represent modifications of the same protein subunit. The three weak spots, labelled by arrows in Fig.l, were present in all preparations

of MCP.

FIGURE 1. Two-Dimensional Gel Electrophoresis of Human MCP. The subunits, stained with Coomassie Blue, have been designated spots are marked by arrows.

as

shown.

Minor

hybridomas producing antibodies to MCP. Their subunit 1. determined by immunoblotting of gels like that in Fig. Four of the antibodies reacted with the same subunit, so only 4 different reactivities were obtained. These are shown in Fig. 2. Antibodies MCP2 and MCP21 (Fig. 2,a and c) reacted with a protein subunit and with a minor spot of protein with lower isoelectric point than the reactive subunit but the same Mr. The minor immunoreactive spots are not visible on blots stained for protein with Ponceau S. However, they seem to coincide with two of the three weak spots in gels stained with the more sensitive Coomassie Blue, and labelled with arrows in Fig. 1. All the antibodies reacted equally well in ELISA with native MCP as with MCP that had been denatured in 6 M urea before adsorption to the plates (results not shown). None of the antibodies bound to lobster MCP (17, results not shown) but several cross-reacted with MCP from rat: (18) or rabbit tissues. These results and isotypes are summarised in Table 1, We tested if one. antibody interferes with the binding of another. The antibodies were diluted to well within the plateau region of their ELISA titration curves and were added either alone or in pairs to microtiter plates with adsorbed MCP. The total amount af antibody bound is shown in Table 2. The results indicate that 3 of the antibodies that react with subunit 7 on blots (i.e. MCP2, MCP16, and MCP20) also compete for binding to MCP adsorbed to microtiter plates. Therefore, it is likely that these antibodies have identical or closely spaced epitopes, whereas MCP28 has a distinct epitope on the same subunit. We

obtained

specificities

7

were

510

I, o o

o

o °

..

o

FIGURE 2. Reactivity of Monoclonal Antibodies with Human MCP. The MCP subunits were separated as in Fig. 1 and blotted onto nitrocellulose. Immunoreactive spots are marked by arrows. Other protein spots are outlined. The antibodies were: a, MCP2 (MCP16, MCP20, and MCP28 gave identical results) ; b, MCP17; c, MCP21; d, MCP25. The monoclonal antibodies do not affect the proteolytic activity of MCP. MCP was incubated for 2 h with a 24-fold molar surplus of each of the monoclonal antibodies or with 2 polyclonal rabbit antibodies to human MCP. This treatment activities inhibited the toward hydrolytic succinyl-ala-phe-phemethylcoumarylamide and benzyloxocarbonyl-ala-arg-arg-methylcoumarylamide by about 10 %. However, similar results were obtained with a monoclonal antibody with an irrelevant specificity, as well as with preimmune rabbit IgG (results None of the antibodies can therefore neutralize the hydrolytic not shown). activity of MCP in a specific way, in agreement with Rivett (19, 20).

TABLE 1 Monoclonal Antibodies to Human MCP Subunit designations are shown in Fig 1. Cross reactivities were determined by ELISA of undiluted hybridomas culture supernatants and are expressed as the absorbances in wells coated with rat or rabbit MCP relative to that obtained with human MCP. Monoclonal

antibody MCP2 MCP 16 MCP 17 MCP20 MCP21 MCP25 MCP28

Isotype

IgG] IgG] IgG] IgG,

IgG] IgG2b IgG,

Subuni t

specificity 7 7 9 7

11 12 7

511

Cross Rat

5 24 28 12 0 0 6

reactivity (%) Rabbit 106 107 58 103

102 10 1.00

TABLE 2

Binding-Competition Assay

of Monoclonal Antibodies to Human MCP.

The

competition between antibodies Saturating amounts of individual

binding to MCP was studied with ELISA. antibodies and pairs of antibodies were incubated with MCP adsorbed to microtiter plates and the total amount of antibody bound was determined with peroxidase-conjugated second antibody. The binding is expressed relative to the binding of individual antibodies, which are taken as 1, as explained in methods^. Ideally, independent binding should give results 2, whereas strong competition should give, binding = 1. Data are means 3. 1 S.E.M., N for

=

-

Antibody #

1.00±0.01

// 2

#16

#17

1120

#21

1.00+0.02

1.90+0.02 1.96±0.02 1.00+0.04

0.99+0.0.1 1.0210.01 1.96+0.01 1.00+0.01

1.33±0.01 1.43±0.01 1.65+0.03 1.48±0.03 1.0010.01

2

1.00+0.01

H 16 il 17 il 20 il 21 il 25 il 28

il 25 1.66+0.02 1.83±0.02 2.37+0.06 1.80+0.02 1.66+0.02 1.00+0.01

il 28 1.56+0.02 1.56±0.01 1.87+0.03 1.5810.01 1.31±0.01 1.75+0.03 1.0010.02

the antibodies that react with different subunits, namely MCP17, and MCP25, were purified and coupled to Sepharose for use in immunoprecipitations. Initially all 4 immobilized antibodies bound soluble MCP, but MCP17 lost binding capacity within a few days. Fig. 3 shows that with each of the remaining 3 antibodies, the electrophoretic patterns of MCP precipitated from HeLa cells, kidney, placenta, and liver were very similar or identical. The precipitates from placenta, and in particular from HeLa cells, contained a number of proteins in excess of MCP. Several of these proteins may be components of the 26 S proteinase. The proteins were not precipitated from liver and kidney, perhaps because these tissues, obtained by autopsy, had been deprived of ATP, so that the 26 S proteinase could dissociate into MCP and its other Four

MCP20,

of

MCP21,

components (7, 8).

Blots of MCP were probed for the presence of glycoproteins. Biotinylated lectins of the following specificities (21) were used: Canavalia ensiformes, Lens culinaris and Pisum sativum (a-D-Man and a-D-Glc); Arachis hypogaea (ß-D-

Gal-(l,3)-D--GalNAc); Glycine max (D-GalNAc); Triticum vulgaris ((ß(l,4)-DGlcNAc)2 and NeuNAc), and Griffonia simplicifolia II agglutinin (ß-D-GlcNAc). None of the lectins reacted with any of the MCP subunits. However, 2 high molecular mass impurities, indétectable by Coomassie blue staining of PAGE gels, if reacted with Canavalia, Lens, and Pisum lectins. This reaction was absent, the

incubation with lectin had been made in the presence of 0.5 M of either

D-

glucose or methyl-o-D-mannoside. Thus, these trace impurities served as internal positive controls for the procedure. The detection of glycoproteins by an alternative method (16) also failed. Therefore, the human MCP seems not to be a glycoprotein. DISCUSSION on the number of subunits in MCP. The presence of subunits has been proposed (20, 22-25). Our 2-D gels showed 13 (in some preparations 14) major spots plus 3 minor ones. The antibodies reactive, with subunits 7 and 10, respectively, each stained a small spots in 2D gels, in addition to the subunit. Since the proteins in these minor spots have, the same Mr but lower pi than the immnnoreactive subunit,

There is no between 12 and 25

consensus

512

FIGURE 3.

Electrophoresis

of MCP Immunoprecipitated from

Human

Tissues

and

Cells. Each of

the monoclonal antibodies MCP20, MCP21, and MCP25 were coupled to and used to precipitate MCP from cell and tissue extracts. Proteins eluted from precipitates (lanes 1 12) or from antibody-coupled Sepharose alone (lanes 14 16) were separated by SDS gel electrophoresis. The extracts were from HeLa cells (lanes 1-3); kidney (lanes 4-6); placenta (lanes 7-9), or liver (lanes 10 12). Lane 13 contains purified placenta MCP as a standard. Antibody MCP20 was used in lanes 1,4,7,10, and 14; MCP21 in lanes 2, 5, 8, 11, and 15; MCP25 in lanes 3, 6, 9, 12, and 16. Immunoglobulin heavy and light chains are indicated by HC and LC, respectively. Positions of Mr marker proteins are shown to the left.

Sepharose

-

-

-

and are present in much lower amounts, we believe them to be traces of subunits that have been deamidated or otherwise modified, either in vivo or during protein purification^ The proteins in spots 3 and 3' (Fig. 1) have the same Mr but. vary in relative amounts. Their combined staining intensity seems to be constant, so they may represent modifications of the same subunit. If so, the MCP is likely to consist of only the 12 subunits numbered in Fig. 1. From electron microscopical studies, Kopp et al. (2) proposed a model whereby the MCP particle consists of 24 subunits arranged in 4 stacked rings. It is therefore that the. MCP particle, comprises 2 of each of the 12 to assume tempting subunits. However, further data are needed to resolve the. matter. Most of the individual subunits in MCP have unique structures (22-26). However, amino acid sequences determined from these proteins (24, 26) or deduced from gene structures (27-29) have shown that there are amino acid sequence homologi.es between several of the subunits. Since the antibodies raised against human MCP and described here reacted with only one subunit each, the epitopes for the 7 antibodies most probably reside outside of such homology regions. Subunit 7 seems to be immunodominant since 4 out of 7 antibodies reacted

513

that subunit. Others have also obtained polyclonal (30, 31) or monoclonal (32, 33) antibodies that react with this subunit. Though raised against the human enzyme, several of the antibodies react with rabbit and rat MCP as well. Such inter-species cross reactivity seems to be common for both polyclonal and monoclonal antibodies to MCP (32, 34, 35). Some antibodies to mammalian MCI1 even react with MOP from yeast (31), and with a similar enzyme from an archebacterium (36). MCP, therefore, is apparently an evolutionary highly conserved protein. However, several of the subunits in rat MCP are reported to be glycoproteins (20, 37) whereas the Drosophila (38) and with

human MCPs are not. It has been suggested that MCP within a single individual is structurally heterogeneous and mny form a family of related but nonidentlcal particles (32, 39). In our experiment the gel electrophoresis patterns of MCP precipitated from extracts of 4 different human tissues or cells with antibodies specific for 3 different subunits were identical or at least very similar. MCP containing these major subunits therefore apparently constitute a homogeneous population of particles with invariant subunit composition. However, Haass and Kloetzel (38) and Ahn et ni. (40) have described minor changes in subunit composition of MCP during larval development, of Drosophila and in embryonic chick muscle,

respectively.

One of the antibodies proved useful for purification of MCP in its (11). Using this MCP as antigen, we are currently developing more monoclonal antibodies that react with MCP subunits. When applied in state

native murlne immuno

electron microscopy and combined with protein cross-linking experiments, such a set of antibodies will be useful in further structural and functional studies of the MCP.

ACKNOWLEDGMENTS

study was supported by the Carlsberg Foundation. Mrs. Anne-Marie B. Mrs. Dorte Kolding, and Mrs. Karen Dissing provided skilled technical assistance. Dr. Burkhardt Dahlmann and Dr. Donald Mykles kindly sent us samples of MCP. Placentae were provided by the Department of Obstetrics and Gynecology, The University Hospital, Copenhagen. This

Lauridsen,

REFERENCES

S., and Orlowki, M. (1980). Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme. .1. Neurochem. 25, 1172-1182

1.

Wilk,

2.

Kopp,

F., Steiner, R., Dahlmann, B., Kuehn, I., and Reinauer, H. shape of the multicatalytic proteinase from rat skeletal Biochim. Biophys. Acta. 872, 253-260 Size

and

(1986). muscle.

B., Pereira, M.E., and Wilk, S. (1991). Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis. J. Biol. Chem. 266,17396-17400

3.

Yu,

4.

Hough, R.F., Pratt, G.W., and Rechsteiner, M. (1988). Ubiquitin/ATPdependent protease, in Ubiquitin (Rechsteiner, M., ed.), pp. 101-134. Plenum Press, Hew York.

5.

Orlowski, M. (1990). The multicatalytic proteinase complex, a extralysosomal proteolytic system. Biochemistry USA. 29, 10289-10297

6.

and Rechsteiner, M. Hough, R.,Pratt,0., (1986). Ublquitin-lysozyme conjugates: identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. J. Biol. Chem. 261, 2400-2408

514

major

7.

Eytan, E., Ganoth, D., Armon, T., and Hershko, A. (1989). ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin. Proc. Nati. Acad. Sei. USA. 86, 7751-7755

8.

Driscoll, J., and Goldberg, A.L. (1990). The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins. J. Biol. Chem. 265, 4789-4792

9.

G.N., McCullough, M.L., Reckelhoff, J.F., Croall, D.E., Ciechanover, A., and McGuire, M.J. (1991). ATP-Stimulated degradation of endogenous proteins in cell-free extracts of BHK21/cl3 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins. Biochim. Biophys. Acta. 1073, 299-308

10.

Tanaka, K., li, K., Ichihara, A., Waxman, L., and Goldberg, A.L. (1986). A high molecular weight protease in the cytosol of rat liver. 1. Purification, enzymological properties, and tissue distribution. J. Biol. Chem. 261,

DeMartino,

15197-15203

11.

Hendil, K.B., and Uerkvitz, W. (1991). The human multicatalytic proteinase: affinity purification using a monoclonal antibody. J. Biochem. Biophys. Meth. 22, 159-165

12.

Harlow, E. and Lane, D. (1988). Antibodies: a laboratory Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

13.

Gherardi,

14.

E., Hutchings, A., Galfré, G. and Bowyer, D.E. monoclonal antibodies to rabbit and human serum low-density Biochem. J. 252, 237-245

O'Farrell, proteins.

15.

P.H.

manual, (1988).

Cold

Rat

lipoprotein.

(1975). High resolution, two-dimensional electrophoresLs 250, 4007-4021

of

J. Biol. Chem.

O.J. S., Lihme, A.O.F., B0g-Hansen, T.C. and Bjerrum. (1985). Lectin blotting of normal and derivatised membrane proteins, in Lectins, Biology, Biochemistry, Clinical Biochemistry (BçSg-Hansen, T.C. & Breborowicz, J. , eds. ) vol. 4, pp.241-252, W. de.Gruyter, Berlin.

Naaby-Hansen,

,

16.

Gershoni, J.M., Bayer, E.A., and Wilchek, M. (1985). a novel reagent for glycoconjugates: enzyme-hydrazide aldehydes. Anal. Biochem. 146, 59-63

Blot analyses the detection

of of

-

17.

Mykles, D.L. (1989). Purification and characterization of a multicatalytic proteinase from crustacean muscle: comparison of latent and heat-activated forms. Arch. Biochem. Biophys. 274, 216-228

18.

Dahlmann,

19.

Rivett,

20.

Rivett, A.J., multicatalytic

21.

Wu, A.M., Sugii, S., and Herp, A. (1988). A table of lectin carbohydrate Lectins. Clinical specificities, in Biology, Biochemistry, Biochemistry. ( B0g-Hansen,T.C. & Freed,D.L.J., eds.), vol. 6, pp.723-740. Sigma Chemical Company, St.Louis, MO

B. , Kuehn, I,., Rutschmann, M., and Reinauer, H. (1985). Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle. Biochem J. 228, 161-170

A.J. (1989). The multicatalytic activities. J.B.iol. Chem. 264, 12215-12219

proteinase.

and Sweeney, S.T. (1991). Properties proteinase complex revealed by the use. antibodies. Biochem. J. 278, 171-177

515

Multiple

catalytic

of subunits of the of subunit-spec!fie

22.

Grainger, J.I.., and Winkler, M.M. (1989). The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs. I. Cell Biol. 109, 675-683

23.

Kleinschmidt, .I.A., Hiigle, B. Grund, C. and Franke, W.W. (1983). The cylinder particles of Xenopus leavis. Eur. J. Cell Biol. 32, 143-156

24.

Lee, L.W., Moomaw, C.R., Orth, K., McGuire, M.J., Slaughter, C.A. (1990). Relationships among the molecular weight: proteinase, macropain (proteasome).

,

,

DeMartino, subunits Biochim.

1073, 178-185

G.N.,

of

the

22S

and

high

Biophys. Acta.

25.

Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, Y., Sato, M., Tanaka, N., Katsube, Y, Kameyama, K., and Takagi, T. (1988). Molecular organization of a high molecular weight multi-protease complex from rat liver. J. Mol. Biol. 2.03, 985-996

26.

Lilley,

sequence

similarities

complex.

FEBS

K.S., Davison, M. D., and Rivett, A..I. (1990). N-Termina] between components of the multicatalytic proteinase left. 262, 327-329

27.

Haass, C, Pesold-Hurt, B., Multhaup, G., Beyreuter, K., and Kloetzel, P.-M. (1990). The Drosophila PROS-28.1 gene is a member of the. proteasome gene family. Gene. 90, 235-241

28.

Sorimachi, H., Tsukahara, T., Kawasaki, H., Ishiura, S., Emori, Y., Sugita, H., and Suzuki, K. (1990). Molecular cloning of cDNAs for two subunits of rat multicatalytic proteinase. Evidence of N-terminal conserved and C-terminal diverged sequences among subunits. Eur. J. Biochem. 193,

775-781

29.

Tamura, T., Lee, D.H., Osaka, F., Fujiwnra, T., Shin, S., Chung, C.H., Tanaka, K., and Ichihara, A. (1991). Molecular cloning and sequence of of cDNAs for five major subunits human analysis proteasomes (multicatalytic proteinase complexes). Bior.him. Biophys. Act n 1089, 95-102

30.

Hügle, B., particles

31.

Kleinschmidt, .I.A., Escher, C, and Wolf, D.H. (1988). Proteinase yscE of yeast shows homology with the 20S cylinder particles of Xenopus Jaeyis. FEBS

Kleinschmidt, J.A., and Franke, W.W. (1983). The 22S cylinder of Xenopus laevis II. characterization and Tmmunological localization of their proteins in tissues and cultured cells. Europ. J. Cell Biol. 32, 157-163

lett.

239,

32. Grossi

Pal,

de

J.K.,

prosomes

as

35-40

Sa, H.-F., Martins de Sa, C, Harper, F., Coux, 0., Akhayat, 0., Florentin, Y., and Scherrer, K. (1988). Cyto localization of a

function of differentiation. .1. Cell Sei. 89,

151-165

33. Kumatori,

A., Tnnakn, K., Inamnra, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawc'i, T., Sliin, S., and Ichihara, A. (1990). Abnormally high expression of

proteasomes in human le.ukemic cells. Proc. Nati. Acad. Sri. USA 87, 7071-70 75

34.

Falkenburg, P.-C, Haass, C. Kloetzel, P.-M., Riedel, B. Kopp, F., Kuehn, L. and Dahlmann, B. (1988). Drosophila small rytoplasmic 19S rjbonucleoprotein is homologous to the rat multicatalytic proteinase. Nature. 331, 190-192.

35.

Tanaka, K., Yoshimura, T., Kumatori, A., Ichihara, A., Ikai, A., Nishigai, M., Kameyama, K., and Takagi, T. (1988). Proteasomes (multi-protease complexes)

,

,

516

,

20S ring-shaped particles in as Chem. 263, 16209-16217

a

variety

of

eukaryotic cells.

J.

Biol.

36.

Dahlmann, B., Kopp, F., Kuehn, L., Niedel, B., Pfeifer, G., Heger], R., and Baumeister, W. (1989).The. multicatalytic proteinase (prosome) is ubiquitous from eukaryot.es to archebacteria. FEBS left. 251, 125-131

37.

Tomek, W., Adam, G., and Schmid, H.-P. (1988). Prosomes, small RNP particles, contain glycoproteins. FEBS Lett. 239, 155-158

38.

Haass,

changes

C,

and Kloetzel, P.M. its subunit pattern

in

(1989). Drosophila proteasome during development. Exptl. Cell

cytoplasmic

undergoes Res.

180,

243-252 39.

P.-F..,

Falkenburg,

characterization

of

and three

Kloetzel, different,

multicatalytic proteinase (proteasome). 40.

P.M.

(1989). subpopulations

Identification and the Drosophila J. Biol. Chem. 264, 6660-6666. of

Ahn, J.Y., Hong, S.O., Kwak, K.B., Kang, S.S., Tanaka, K., Ichihara, A., Ha, D.B., and Chung, C.H. (1991). Developmental Regulation of Proteolytic Activities and Subunit Pattern of the 20S Proteasome in Chick Embryonic Muscle. J. Biol. Chem. 266, 15746-15749 Address

reprint requests

Klavs B. Hendil

August Krogh Institute 13, Universitetsparken DK-2100 Copenhagen 0. Denmark

publication: 4/21/92

Received for

Accepted:

3/10/92

517

to: