0019-9567/78/0019-0272$02.00/0 INFECTION AND IMMUNrrY, Jan. 1978, p. 272-280 Copyright © 1978 American Society for Microbiology

Vol. 19, No. 1 Printed in U.S.A.

Changes in Liver and L-Cell Plasma Membranes During Infection with Coxiella burnetii N. MARECKI,' F. BECKER,'t 0. G. BACA,2 AND D. PARETSKYI* Department of Microbiology, University of Kansas, Lawrence, Kansas 66045,' and Department of Biology, University of New Mexico, Albuquerque, New Mexico 871312

Received for publication 13 June 1977

Changes in plasma membrane proteins of guinea pig liver and L-929 cells were studied during infection with Coxiella burnetii. Polypeptide species resolved by disc polyacrylamide gel electrophoresis with sodium dodecyl sulfate showed quantitative but no qualitative differences between uninfected and infected samples. When the O'Farrell technique of isoelectric focusing, followed by sodium dodecyl sulfate-slab gel polyacrylamide gel electrophoresis, was employed, additional polypeptides were resolved. Livers and L cells were labeled with [3H]glucosamine. Infected livers incorporated less [3H]glucosamine in the membrane proteins than uninfected material, presumably indicating lower glycoprotein levels. Infected L-cell membranes incorporated greater amounts of [3H]glucosamine, and also were labeled to a greater extent than uninfected membranes, employing the [251I]lactoperoxidase technique. Uninfected L cells showed a greater agglutinability with concanavalin A than did infected cells. Infected livers had much greater levels of cyclic adenosine 3',5'-monophosphate. The data indicate changes in plasma membranes as a result of infection. Possible physiological consequences of membrane changes are discussed.

Among the pathophysiological events in guinea pigs experimentally infected with the rickettsial agent of Q fever, Coxiella burnetii, is the characteristic development of a fatty liver (4, 27, 28). Fatty liver genesis may be due to a variety of events, including impaired lipid export because of defective membrane permeability (4, 22). C. burnetii lipopolysaccharide itself induces fatty livers (3), reminiscent of the altered permeability of intestinal cells (35), and lipid release from adipocytes by cholera toxin (18). Furthermore, L cells infected with C. burnetii display altered permeability characteristics to vital dyes (J. Stueckemann and D. Paretsky, unpublished data). Because the plasma membrane plays a key role in cell growth, metabolism (19, 31), and pathology, the plasma membranes of liver and L cells infected with C. burnetii were studied. MATERIALS AND METHODS Organisms, cells, and animals. Male Hartley guinea pigs, 250 to 350 g (Hilltop Laboratory Animals, Inc., Scottsdale, Pa.) were infected with phase I C. burnetii, Nine Mile strain, by intraperitoneal inoculation with 0.5 ml of a 20% yolk sac suspension in 0.1% skim milk-0.05 M potassium phosphate buffer (pH 7.4), 50% lethal dose 10-5-. L-929 cells adapted to suspension culture were originally obtained from R. tPresent address: Stanford Research Institute, Menlo Park, CA 94025.

Erickson, University of Colorado Medical School, Denver. The cells were maintained in suspension culture in an antibiotic-free, spinner-modified Eagle minimal essential medium (Schwarz/Mann, Orangeburg, N.Y.), supplemented with 5% heat-inactivated calf serum (K. C. Biological, Inc., Lenexa, Kans.) and 0.1% methylcellulose (15 centipoises; Fisher Scientific Co., St. Louis, Mo.). Cell suspension (50 ml) was incubated in a 250-ml screw-cap Erlenmeyer flask in an incubator-rotary shaker at 35°C, 80 to 100 rpm. Cell cultures were divided three times weekly, and the newly inoculated medium contained about 2.5 x 105 cells per ml. A persistent infection in L cells was established with phase I C. burnetii (J. Stueckemann and D. Paretsky, unpublished data). Infected L cells contained 10; to 104 rickettsiae per cell; the population was 90 to 95% infected. Infected L-cell diameters were 50% greater than those of uninfected cells (J. Stueckemann and D. Paretsky, unpublished data). Infected and uninfected L cells in the log phase of growth were employed. Liver plasma membranes. Plasma membranes were isolated by the methods of Neville (24) and Chandrasekhara and Narayan (8). Guinea pigs were decapitated 84 h postinfection and exsanguinated, and the livers and spleens were rapidly removed and weighed. Impression smears stained by the Giminez method showed the organs to be 3+ to 4+ at this time. The livers were homogenized in 10 to 15 ml of 1 mM NaHCO3 (pH 7.5) per liver, employing a Tekmar Tissumizer (Tekmar, Cincinnati, Ohio). The density of the sucrose solutions, critical for obtaining reproducible results, was monitored with an Abbe refrac-

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tometer. Homogenate suspensions in 1.197 d sucrose (final) were overlaid with 17 ml of 1.18 d sucrose, which in turn was overlaid with 10 ml of 1.16 d sucrose, and centrifuged in an SW27 rotor at 130,000 x g for 2 h. The membranes were collected from the 1.16/1.18 d interface, suspended in 1 mM NaHCO3, and centrifuged in the SW27 rotor at 113,000 x g for 15 min. The washed, pelleted membranes were resuspended in 2 to 3 ml of 1 mM NaHCO3 and either used immediately or stored at -20°C until use. L-cell plasma membranes. L-cell plasma membranes were prepared by the methods of Atkinson and Summers (2) and Brunette and Till (6). Cells were harvested, washed twice with cold 0.15 M NaCl, and disrupted in a type B Dounce homogenizer. The debris was removed by centrifuging at 1,000 x g, and the membrane in the supernatant was separated on discontinuous sucrose gradients, 39/50% (wt/wt) (15). Membrane preparations were assayed for purity employing enzyme activities as markers. Glucose-6-phosphatase (14) and Na+- and K+-dependent adenosine triphosphatase (6) and adenosine 5'-monophosphate nucleotidase were measured by release of inorganic phosphate from the respective substrates. Inorganic phosphate was measured colorimetrically (9). Cholesterol and phospholipid content may additionally serve as distinguishing characteristics of membranes (38). Lipids were extracted from unfractionated cell homogenates and from their plasma membranes (38), and the phospholipid and cholesterol contents (1, 5) were measured. Proteins were measured by the methods of Lowry et al. (20). Isotope labeling. L-cell membranes labeled with [3H]glucosamine were prepared by incubating 50 ml of a cell suspension containing 2.5 x 105 cells per ml for 40 h with 100 ,uCi of D-[6-3H]glucosamine (10.1 Ci per mmol; New England Nuclear Corp., Boston, Mass.). Total incorporation of [3H]glucosamine into intact cells was measured in a sample of cells precipitated with trichloroacetic acid. Incorporation into peptide species was analyzed in membranes isolated from the L cells. L cells were labeled with i1I (29) employing carrier-free NainI (16.2 Ci per mg, Schwarz/Mann), lactoperoxidase (122 IU per mg; Calbiochem, La Jolla, Calif.), and H202. Incorporation was analyzed in membranes isolated from these cells. Liver plasma membranes were labeled with glucosamine by injecting guinea pigs intraperitoneally with 250 ,ICi of D-[1-3H]glucosamine (320 mCi/mmol) per animal, together with 0.5 ml of lx Eagle minimum essential medium amino acid mixture, 3 h before sac-

rifice. PAGE. Polyacrylamide gel electrophoresis (PAGE) was conducted essentially by the methods of Neville (24), employing a 5% acrylamide analytical gel 10 cm long, with a 3.2% stacking gel. The gel buffer was 0.5 M tris(hydroxymethyl)aminomethane (pH 8.9). After polymerization, gels were prerun for 45 to 60 min at 1.5 mA per gel. The lower reservoir buffer was 0.42 M tris(hydroxymethyl)aminomethane (pH 9.2), and the upper reservoir buffer was 0.04 M boric acid-0.04 M tris(hydroxymethyl)aminomethane-0.1% sodium dodecyl sulfate (SDS) (pH 8.6). Membrane preparations were first solubilized with 2% SDS and 10% ,Bmercaptoethanol (final concentrations). The gels were

273

loaded with 5 pl of 0.1% bromophenol blue as a front marker, and then with 10 to 20,ul of sample containing 25 to 35,ug of membrane protein. Gels with molecular weight markers were prepared for simultaneous electrophoresis. Electrophoresis was conducted for 30 min at 0.5 mA per gel, after which the current was increased to 1.5 mA per gel, and continued until the dye front was 5 mm fron the end of the gel. Gels were removed and stained overnight with 0.05% Coomassie brilliant blue (R-250, Sigma), in 45.4% methanol-9.2% glacial acetic acid. The gels were destained electrolytically in 7.5% glacial acetic acid-5% methanol and scanned at 600 nm in a Gilford 2400 spectrophotometer equipped with a gel scanner. After absorbancy profiles were obtained, radioactive gels were frozen at -70°C and cut into 1-mm slices. Gels slices with [3H]glucosamine-labeled membranes were digested overnight with 30% H202 at 37°C, and the radioactivity was measured in a Tri-Carb liquid scintillation spectrometer. Gel slices containing '25I-labeled membranes were measured in a Nuclear-Chicago gamma counter. L-cell membrane proteins were also separated by two-dimensional (2-D) slab gel chromatography (25). Proteins were suspended in 25 pi of 0.01 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) and mixed with 25 1l of "lysis buffer A" (25). The final concentrations were 2 ig of protein per pl; 0.8% Ampholine (LKB Instruments, Rockville, Md.), pH 5 to 8; 0.2% Ampholine, pH 3 to 10; 2.5%,B-mercaptoethanol, and 9.5 M urea. The sample was layered on isoelectric focusing gels and subjected to electrophoresis at a constant voltage of 400 V for 18.5 h through a 10.5-cm gel path. The resultant pH gradient was 4.7 to 6.8 and linear to 8.5 cm. The gels were then equilibrated for 30 min in the SDS sample buffer (25) and then subjected to electrophoresis in the second dimension through a 13-cm gel path, at 20 mA, for about 5 h or until the marker dye was 5 mm from the gel edge. The gel slab was stained in 0.1% Coomassie blue R-250-50% methanol-7.5% acetic acid, decolorized, and dried.

RESULTS Membrane preparations. Plasma membrane preparations were evaluated by various criteria for purity and homogenity. Phase contrast microscopy showed intact membranes and large membrane fragments free of nuclei. Stained plasma membrane preparations from infected L cells revealed the presence of a few C. burnetii. Intact C. burnetii demonstrate little or no enzymatic activity (26), and solubilization of proteins from the rickettsiae does not occur under the conditions here employed (J. Stueckemann and D. Paretsky, unpublished data). Glucose-6-phosphatase activity, used as an indicator of substantial contamination of plasma membranes by intracellular organelles (33), showed no increase of specific activity in either liver or L-cell membrane preparations (Table 1). In contrast, plasma membrane-associated Na+-K+-dependent adenosine triphosphatase activity increased 2.6-fold. Similarly, 5'-nucleotidase

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INFECT. IMMUN.

MARECKI ET AL. TABLE 1. Criteria for plasma membrane purity Activity a Marker

Glucose-6-phos-

Tissue

Sample

Liver

Homogenate Membrane Hom/Mem Homogenate Membrane Hom/Mem

2.72 3.92 1.4 0.19 0.23

1.2

3.02 4.83 1.6 0.15 0.19 1.3

phatase L cell

Control ± 0.08 ± 0.34 ± 0.02 ± 0.03

Infected + 0.25 ± 0.21 ± 0.02 ± 0.02

5'-Nucleotidase

Liver

Homogenate Membrane Hom/Mem

0.53 ± 0.01 1.48 ± 0.20 2.8

0.49 ± 0.05 1.69 ± 0.08 3.4

Na+-K+-adenosine triphosphatase

L cell

Homogenate Membrane Hom/Mem

0.64 ± 0.12 1.66 ± 0.18 2.6

0.89 ± 0.20 2.35 ± 0.26 2.6

Liver 0.59 ± 0.05 0.43 ± 0.01 Membrane L cell Membrane 0.65 ± 0.04 0.05 ± 0.02 aActivity as micromoles of inorganic phosphate released per milligram of protein per hour, ± standard error of the mean. ' Ratio as micromoles per milligram of protein.

Cholesterol/ phospholipidb

(adenosine 5'-monophosphate -- adenosine + inorganic phosphate), which is associated with the plasma membrane (10), was enriched about threefold. Cholesterol-phospholipid ratios serve as additional distinguishing characteristics of plasma membranes. Plasma membrane fractions of eucaryotic cells have cholesterol-phospholipid ratios of greater than 0.5, and intracellular organelles such as the endoplasmic reticulum and mitochondrial membranes have cholesterolphospholipid ratios of less than 0.1 (10, 32, 38). The plasma membrane preparations from uninfected liver and L cells had cholesterolphospholipid ratios of 0.59 and 0.65 (Table 1). The lower ratios from corresponding infected material were due to increased phospholipid content, correlating with the findings in earlier work (4). The data in Table 1 are consistent with those of other reports (10, 32, 38), indicating a high degree of purity of plasma membrane preparations. It may be of some interest to note that the actual yield of plasma membranes from infected livers was about 60% that from uninfected livers (0.89 + 0.13 versus 0.51 ± 0.06 mg of protein per g of liver). Infected livers had 18.1 + 3.6 nmol of cyclic adenosine 5'-monophosphate per liver, compared with 8.4 ± 0.6 nmol per uninfected liver; or 6.5 + 1.5 pmol per mg of protein, infected, versus 4.1 + 0.2 pmol per mg of protein, uninfected. PAGE. Disc PAGE of plasma membranes from uninfected and infected L cells showed qualitatively similar polypeptide profiles (Fig. 1). About 37 polypeptide species with molecular

weights between 250,000 and 15,000 were resolved. The major species were between 100,000 and 67,000 daltons for infected and uninfected membrane preparations. Similar patterns were obtained for both uninfected and infected L-cell membrane preparations, but there were quantitative differences between given species. Analogous results were obtained for plasma membranes from uninfected and infected livers (Fig. 2; Table 2). Although disc SDS-PAGE separates polypeptides of different molecular weights, chemically different species with the same molecular weights may show the same gel migration. On the other hand, 2-D electrophoresis, which also employs isoelectric focusing, effects separation of such similar-molecular-weight species on the basis of charge difference not possible by conventional disc PAGE (25). Such a 2-D analysis did indeed reveal differences between the polypeptides of L-cell infected and uninfected membranes (Fig. 3). Some 10 to 15 additional peptides were resolved in the 2-D procedures, which are being currently employed in our further studies on membrane compositions during infection. These experiments wiRl couple 2-D chromatography with autoradiography. [3H]Glucosamine-labeled cells. Plasma membranes were isolated from L cells incubated with D-[3H]glucosamine for 40 h. Membranes from uninfected cells incorporated 6.7 ± 0.3 x 105 dpm per mg of protein, whereas infected cell membranes had 8.3 + 0.05 dpm per mg of protein, an increased incorporation of 25%. The polypeptide profile of glucosamine-la-

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PLASMA MEMBRANE CHANGES DURING INFECTION

275

Front MOL HI. X 10-4 FIG. 1. SDS-PAGE patterns of L-cell plasma membranes. Samples were solubilized in 2% SDS and 10%0 ,8-mercaptoethanol; 30 pg of membrane protein was loaded on each 5%, 10-cm gel. After electrophoresis, gels ) Uninfected; (----- ) infected. were stained and decolorized, as described in text, and scanned at 600 nm. (

beled membranes on PAGE (Fig. 4) was similar to that in Fig. 1. In both infected and uninfected cell membranes, the major labeled species were between 60,000 and 170,000 daltons (Fig. 4). Approximately 50% of the label was incorporated into polypeptide species between 67,000 and 150,000 daltons. Again, only quantitative changes in polypeptide species of infected cell membranes were observed. The increase in incorporation by infected cell membranes was substantially different for polypeptides between 25,000 and 200,000 daltons. Livers also were labeled with D-[3H]glucosamine. Infected liver plasma membranes had incorporated 2,400 ± 20 dpm per mg of protein, or about 51% that of uninfected membranes (4, 730 ± 30 dpm per mg of protein). Unfortunately, the levels of radioactivity of the individual peptides resolved by PAGE were too low to present a clear pattern. I25I-labeled membranes. To study only surface proteins, intact nonnal and infected L cells were labeled with "2I using lactoperoxidase and H202. This method selectively labels exposed tyrosine residues (23). Membranes were then

prepared, and the pattem of "2I incorporation was determined (Fig. 5). The polypeptide profile obtained was similar to that of unlabeled cells (Fig. 1). Approximately 30% of the label was incorporated into that region containing the major polypeptide species, that is, 67,000 to 100,000 daltons. A similar pattern of '25I labeling of surface components has been reported (15, 17), although the present system achieved greater resolution. The ratios of labeling for various molecularweight ranges showed more variation among the polypeptide groups than was seen with L-cell glucosamine labeling. There was a substantial reduction in "I labeling of infected cell surfaces in the highest and lowest molecular-weight groups. Also, those groups with the least difference in 125I labeling showed the greatest difference with respect to glucosamine incorporation. DISCUSSION Guinea pigs characteristically develop fatty livers during experimentally induced Q fever, with an accumulation of triglycerides and (to a lesser extent) phospholipids (4, 27).

276

MARECKI ET AL.

INFECT. IMMUN.

lipopolysaccharide into guinea pigs (4). Because the plasma membrane plays a critical role in liver transport, changes in the plasma membrane could be implicated in hepatic steatosis. The evidence in this report supports the concept of changes in liver plasma membrane, including lipid composition as cholesterol-phospholipid ratios, the relative abundance of several polypeptide species, and glucosamine incorporation (glycoprotein synthesis). Similar membrane changes have been reported in other pathologies, such as preneoplastic and neoplastic rat liver (8), and virus-transformed cell lines (13, 21, 37, 39). In light of the pharmacological activity of the C. burnetii lipopolysaccharide, it should be noted that cholera toxin affects the function of plasma membranes of infected cells (11) and induces lipid release from fat cells (35). Because hormones such as cortisol and cyclic adenosine 5'-monophosphate (28) and other humoral factors such as lipases (4) may be implicated in the liver pathobiology of Q fever, arising TABLE 2. SDS-PAGE comparison of infected and uninfected liver plasma membrane proteinsa Peak no. Change' Mol wt x 10-' 1 2 3 4 5 6 7 8

MOL. WT. X 10-3 FIG. 2. SDS-PAGE patterns of liver plasma mem-

branes. Samples were solubilized as in Fig. 1, and 50 ,ug of membrane protein was subjected to electrophoresis on a 7.5% gel. After electrophoresis, gels were analyzed as in Fig. 1. (a) Uninfected; (b) infected.

Whereas fatty changes are a common response of liver to injury, numerous drugs, hepatoma, partial hepatectomy, and a variety of infectious diseases and endotoxins, the causative mechanisms of lipid infiltration and accumulation may vary. One pattern would involve increased lipase activity at the fat depots, resulting in higher levels of serum lipid, followed by liver lipid import, with subsequent defective lipid export (12). We have previously shown that during Q fever lipase activity is increased at the fat depots with increased serum lipid (4), and that these conditions also obtain after injection of C. burnetii

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

-

0 0 0 0 -

0 -

-

0 + + + + 0 -

0 0 + + 0 +

130 130 130 130 130 130 130 130

130 120 105 96 88 82 72 68 64 59 56 52 49 44 42 40 37 34 28 20

a Electrophoresis and profiles shown in Fig. 2. Peak numbers correspond to those in Fig. 2. b Relative to corresponding peaks of uninfected membranes. +, Increase; -, decrease; 0, no change. e Approximate molecular weight of protein species, based on relative mobility.

A-

e

7*45

U ~ ~ ~

pH

~

.05S >

~

00

B

±

FIG. 3. 2-D electrophoresis of L-cell plasma membranes (according to O'Farrell [251, as described in text). (A) Uninfected; (B) infected. First dimension: pH gradient 7.45 -* 4.65, cathode -* anode. Second dimension: gel length, 8.7 cm. 277

278

INFECT. IMMUN.

MARECKI ET AL. 12r

2

5,

3

2

20

15

7 5 MOL wT. X 10-4

10

2

Dye Front

FIG. 4. Distribution of [3H]glucosamine inpolypeptides of L-cell plasma membranes. Labeled membranes were solubilized and subjected to electrophoresis as in Fig. 1. After electrophoresis, gels were frozen, and 1mm slices were made. Gel slices were solubilized in H202 and counted. 10

NORMAL 9

7

~~~~~~~~INFECTED

-I

3 -~~~~~~~~~~~~~~~ 2~

~

~

~

~

7 5 10 20 15 MOL4 WT. X 10-4

2

Dye

FIG. 5. Distribution of "'I in polypeptides of L-cell plasma membranes. Labeled membranes were treated and subjected to electrophoresis as in Fig. I and 4. Radioactivity of the gel slices was measured in a gamma counter. ( ), Uninfected; ( ----- ) infected.

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PLASMA MEMBRANE CHANGES DURING INFECTION

directly as a result of infection (3) or from infection stress, the response of the L cell to infection was studied. L cells in suspension culture were persistently infected with C. burnetii, and the rickettsiae proliferated within a cytoplasmic vacuole. Purified plasma membranes of uninfected and infected L cells were subjected to disc electrophoresis, and, as in the case of liver membranes, the same number of polypeptide species was resolved (Fig. 1). Compared with the liver membranes, there were lesser quantitative amounts of peptide species from infected membranes between 40,000 and 120,000 daltons. Disc PAGE resolved approximately 30 peptide species from liver and L-cell plasma membranes, with no qualitative differences seemingly existent between infected and uninfected material. Plasma membranes from virus-transformed cells also showed an apparent quantitative difference after PAGE and Coomassie blue staining, although the peptide profiles were quite similar (13). The O'Farrell technique of 2-D electrophoresis was employed with L-cell membranes in a further attempt to identify possible effects due to infection. The 2-D procedures separate species on the basis of their isoelectric points and molecular weights (25). Many more peptides were now resolved, with clear differences between infected and uninfected membrane peptides. Some intact rickettsiae were recovered in the plasma membranes from infected cells, but the peptide contribution of the rickettsiae must be considered as negligible, because, even when C. burnetii suspensions were incubated for 1 h in ethylenediaminetetraacetate, subjected to sonic treatment, and boiled for 1 min in 1% SDS and 10% fB-mercaptoethanol, only 4% of rickettsial protein was released. Such conditions are far more rigorous than those used to solubilize the plasma membrane preparations. There is a further possibility that the new peptide species revealed by 2-D PAGE may be constituents of the lysosomal vacuole in which the rickettsiae proliferate, or even components of the vacuolar membrane, which accompanied the plasma membranes during purification. The use of surface-specific probes such as pyridoxal phosphate/NaB3H4 (7, 16), galactose oxidase/KB3H4 (16), and periodate/NaB3H4 (34) together with 2-D PAGE will help clarify the picture. Unlike the liver, infected L-cell membranes incorporated a greater amount of [3H]glucosamine. Similar results were obtained with 125I labeling (Fig. 5). The latter results may be due to the increased surface area of the infected cell, which can be 10 to 40% greater than that of the uninfected cell (J. Stueckemann and D. Paretsky, unpublished data), with resultant greater access of lactoperoxidase to the tyrosine residues on the

279

cell surface and greater iodination. Another index of surface alteration is response to lectins, such as concanavalin A. When 7.5 x 105 L cells per ml were incubated in 25 ,ug of concanavalin A per ml in a plastic petri dish at room temperature for 15 min, uninfected cells aggregated to a greater extent than did infected cells. More uninfected cells formed aggregates, and the aggregates were larger. After 60 min, only 15% of uninfected cells and 20% of infected cells remained as unaggregated, single cells. The data correlate with the concept that, during infection, the plasma membrane of the host becomes altered. The physiological and biochemical properties of plasma membranes have been extensively reviewed (for example, 19, 30, 36). Of special interest in pathobiology are the roles membranes play in the regulation of intracellular processes, receptor site activity, and cell transport regulation. The present report shows that infection with C. burnetii indeed alters the plasma membrane of its host. The data suggest that the development of a fatty liver may be at least in part associated with changes in membrane peptide composition with consequent changes in lipid transport, although this interpretation is as yet unsubstantiated by present evidence. Apparent differences in glucosamine incorporation between infected liver and L-cell membranes may be due to hormonal or other factors to which the organ of the intact aniimal is exposed, or to different metabolisms of glucosamine by liver and L cells. The data indicate the need to establish the identity and function of those membrane peptides that arise during infection, and of such peptides that diminish or disappear. The extent to which the events of Q fever occur in other infections and pathologies should be of interest. ACKNOWLEDGMENTIS We acknowledge the valuable technical assistance of M. L. Anthes. The work was supported by grants from the University of Kansas General Research Fund, the National Science Foundation (PCM76-11706), and the University of New Mexico Research Allocation Committee.

LITERATURE CITED 1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235:769-775. 2. Atkinson, P. H., and D. F. Summers. 1971. Purification and properties of HeLa cell plasma membranes. J. Biol. Chem. 246:5162-5175. 3. Baca, 0. G., and D. Paretaky. 1974. Some physiological and biochemical effects of a Coxiella burneti lipopolysaccharide preparation on guinea pigs. Infect. Immun. 9:939-945. 4. Bernier, R. D., T. Haney, and D. Paretsky. 1974. Changes in lipids of liver and plasma during Q fever. Acta Virol. 18:75-80.

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MARECKI ET AL.

5. Brown, H. A., A. Zlatkis, B. Zek, and A. J. Boyle. 1954. Rapid procedure for the determination of free serum cholesterol. Anal. Chem. 26:397-399. 6. Brunette, D. M., and J. E. Till. 1971. A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5:215-224. 7. Cabantchik, I. Z., M. Balshin, W. Brewer, and A. Rothstein. 1975. Pyridoxal phosphate. An anionic probe for protein amino groups exposed on the outer and inner surfaces of intact human red blood cells. J. Biol. Chem. 250:5130-5136. 8. Chandrasekhara, N., and K. A. Narayan. 1970. Studies on liver plasma membranes of rats fed N-2-fluorenylacetamide. Cancer Res. 30:2876-2880. 9. Chen, P. S., Jr., T. Y. Toribara, and H. Warner. 1956. Micro-determination of phosphorus. Anal. Chem. 28:1756-1758. 10. Coleman, R., and J. B. Finean. 1966. Preparation and properties of isolated plasma membranes from guinea pig tissue. Biochim. Biophys. Acta 125:197-200. 11. Cuatrecasas, P. 1973. Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry 12:3547-3558. 12. Dianzani, M. U. 1973. Biochemical aspects of fatty liver. Biochem. Soc. Trans. 1:903-908. 13. Greenberg, C. S., and M. C. Glick. 1972. Electrophoretic study of the polypeptides from surface membranes of mammalian cells. Biochemistry 11:3680-3685. 14. Harper, A. E. 1965. Glucose-6-phosphatase, p. 788-792. In J. U. Bergmeyer (ed.), Methods in enzymatic analysis. Academic Press Inc., New York. 15. Hubbard, A. L., and Z. A. Cohn. 1975. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J. Cell Biol. 64:438-460. 16. Hunt, R. C., and J. C. Brown. 1974. Surface glycoproteins of mouse L cells. Biochemistry 13:22-28. 17. Hunt, R. C., and J. C. Brown. 1975. Identification of a high molecular weight trans-membrane protein in mouse L cells. J. Mol. Biol. 97:413-422. 18. Kimberg, D. V., M. Field, J. Johnson, A. Henderson, and E. Gershon. 1971. J. Clin. Invest. 50:1218-1230. 19. Korn, E. D. 1969. Current concepts of membrane structure and function. Fed. Proc. 28:6-11. 20. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 21. Meegam E., H. C. Wu, P. H. Black, and P. W. Robbins. 1969. Comparative studies on the carbohydrate-containing membrane components of normal and virustransformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by Sephadex chromatography. Biochemistry 8:2518-2524. 22. Middleton, H. M., III, G. D. Dunn, and S. Schenker. 1975. Alcohol-induced liver injury: pathogenic considerations. In F. F. Becker (ed.), The liver. Normal and abnormal functions. Marcel Dekker Inc., New York.

INFECT. IMMUN. 23. Morrison, M. 1974. The determination of the exposed proteins on membranes by the use of lactoperoxidase. Methods Enzymol. 32:103-109. 24. Neville, D. M., Jr. 1971. Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246:6328-6334. 25. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis ofproteins. J. Biol. Chem. 2 50:4007-402 1. 26. Paretaky, D. 1968. Biochemistry of rickettsiae and their infected hosts, with special reference to Coxiella burneti. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig. 206:284-291. 27. Paretsky, D., C. M. Downs, and C. W. Salmon. 1964. Some biochemical changes in the guinea pig during infectionwith Coxiella burnetii. J. Bacteriol. 88:137-142. 28. Paretsky, D., and J. Stueckemann. 1970. Chemical and biochemical changes in subcellular fractions of guinea pig liver during infection with Coxiella bumneti. J. Bacteriol. 102:334-340. 29. Poduslo, J. F., C. S. Greenberg, and M. C. Glick. 1972. Proteins exposed on the surface of mammalian membranes. Biochemistry 11:2616-2621. 30. Quinn, P. J. 1976. The molecular biology of cell membranes. University Park Press, Baltimore. 31. Singer, S. J. 1974. Molecular biology of cellular membranes with applications to immunology. Adv. Immunol. 19:1-66. 32. Solyom, A., C. J. Lauter, and E. G. Trams. 1972. Plasma membranes from isolated liver cells. Biochim. Biophys. Acta 274:631-637. 33. Swanson, M. A. 1955. Glucose-6-phosphatase from liver. Methods Enzymol. 2:541-543. 34. Van Lenten, L., and G. Ashwell. 1971. Studies on the chemical and enzymatic modification of glycoproteins: a general method for the tritiation of sialic acid-containing glycoproteins. J. Biol. Chem. 246:1889-1894. 35. Vaughan, M., N. F. Pierce, and W. B. Greenough, m. 1970. Stimulation of glycerol production in fat cells by cholera toxin. Nature (London) 226:658-659. 36. Wallach, D. F. H. 1972. The plasma membrane: dynamic perspectives, genetics and pathology. Springer-Verlag, New York. 37. Warren, L., and C. A. Buck. 1976. Chemical changes in neoplastic cell membranes. In L. Bolis, J. F. Hoffman, and A. Leaf (ed.), Membranes and disease. Raven Press, New York. 38. Weinstein, D. B., J. B. Marsh, M. C. Glick, and L. Warren. 1969. Membranes of animal cells. IV. Lipids of the L-cell and its surface membranes. J. Biol. Chem. 244:4103-4111. 39. Wu, H. C., E. Meezam, P. H. Black, and P. W. Robbins. 1969. Comparative studies on the carbohydratecontaining membrane components of normal and virustransformed mouse fibroblasts. I. Glucosamine-labeling patterns in 3T3, spontaneously transformed 3T3, and SV-40-transfonned 3T3 cells. Biochemistry 9:2509-2517.

Changes in liver and L-cell plasma membranes during infection with Coxiella burnetii.

0019-9567/78/0019-0272$02.00/0 INFECTION AND IMMUNrrY, Jan. 1978, p. 272-280 Copyright © 1978 American Society for Microbiology Vol. 19, No. 1 Printe...
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