[77] Cultured cell systems and methods for neurobiology

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

765

plasma membranes of both mouse macrophages and erythrocytes. 22 In the presence of complement, these sera lyse mouse cells whereas in its absence they stimulate pinocytosis and lysosome formation in a dose dependent relationship. Since bovine hemolytic complement is only partially susceptible to 56 ° at 30 minutes, all sera are aged at 4 ° for 2 weeks before use. Fetal calf serum lacks the antimouse antibody and is a less effective inducer of pinocytosis. For routine cultures all sera are employed at a concentration of 20% v/v, but may be increased to as high as 50% for the more rapid formation of lysosomes. Properties of Cultured Mouse Macrophages In the presence of 2 0 - 5 0 % aged calf serum, macrophages do not divide but increase in size and protein content and demonstrate progressive increments in their content of lysosomes 23 and lysosomal hydrolases. Functional properties, such as pinocytosis and phagocytosis, are enhanced, and large amounts of plasma membrane are interiorized to form endocytic vacuoles. The cells become highly oriented on the glass surface, demonstrating a perinuclear area filled with secondary lysosomes or dense granules surrounding a multicentric Golgi apparatus. The peripheral cytoplasm contains long mitochondria extending to the tips of pseudopods and lucent pinocytic vesicles which can be seen moving centripetally toward the perinuclear area. Moderate amounts of rough-surfaced endoplasmic reticulum and occasional polysomes are present. A more detailed account of their properties can be found in review articles. 1~ ~z. A. Cohn and E. Parks, J. Exp. Med. 126, 941 (1967). 2~Z. A. Cohn and M. E. Fedorko, in "Lysosomes in Biology and Pathology" (J. Dingle and H. Fell, eds.), Vol. 1, p. 43. North-Holland Publ., Amsterdam, 1969.

[77]

Cultured

Cell Systems and Methods

for Neurobiology

B y BRUCE K. SCHRIER, SAMUEL n . WILSON, and MARSHALL NIRENBERG

Methods' are described for the culture of cells from the nervous system of the mouse or rat, and for determining the activities of enzymes required for communication between neurons, such as choline acetyltransferase (EC 2.3.1.6); tyrosine hydroxylase (EC 1.14.3a); glutamate decarboxylase (EC 4.1.1.15 ) ; acetylcholinesterase (EC 3.1.1.7), and catechol O-methyltransferase (EC 2.1.1.1). The methods are sufficiently 1S. H. Wilson, B. K. Schrier, J. L. Farber, E. J. Thompson, R. Rosenberg, A. Blume, and M. Nirenberg, J. Biol. Chem. 247, 3159 (1972).

766

ISOLATION AND CULTURE OF CELLS

.r~

I+

++++++~

+

+++++++

++

.t',,c,D

~Q Z 0

•~

o ¢~ t.-*

.~O

8

v ~

N

0

N O

L)

[77]

+

I

[77]

767

CULTURED CELL SYSTEMS, METHODS FOR NEUROBIOLOGY

~)

+++

I

I

~

~

+++~

+

~

I ~

¢,D

I

•

O

°

~ o ~

~

~

~ .~

~.~ ~

~

d~4~nm

~

768

ISOLATION AND CULTURE OF CELLS

[77]

sensitive so that specific activities of most of the above enzymes usually can be determined with protein harvested from one petri dish. Fifty to 100 assays are performed routinely in one day. Cell Lines Properties of clonal lines of mouse neuroblastoma C-1300 are shown in Table I. 2-5 Three types of clones have been obtained with respect to neurotransmitter synthesis: cholinergic clones, adrenergic clones, and clones without appreciable amounts of catechols or acetylcholine2 Each cell line shown in Table I that was examined had acetylcholinesterase activity. Regulatory steps were found for each enzyme; tyrosine hydroxylase, choline acetyltransferase, and acetylcholinesterase specific activities are 40-, 4-, and 25-fold higher, respectively, when homogenates are prepared from stationary rather than logarithmically dividing cells2 -I° Neurites 2,4,11,~2 and excitable membrane activities 13 also respond to regulatory steps; logarithmically dividing cells usually have relatively short processes and passive rather than excitable membranes. A defective neuroblastoma clone ( N I A - 1 0 3 ) without neurites, excitable membrane step A, tyrosine hydroxylase, and choline acetyltransferase has been reported2 ,la The histamine content of an uncloned line of neuroblastoma C-1300 is 17 pmoles per milligram of protein, similar to the level found with mouse brain. Glutamic acid decarboxylase activity was not detected ( < 1%

G. Augusti-Tocco and G. Sato, Proc. Nat. Acad. Sci. U.S. 64, 311 (1969). 3j. B. Olmsted, K. Carlson, R. Klebe, F. Ruddle, and J. Rosenbaum, Proc. Nat. Acad. Sci. U.S. 65, 129 (1970). 4D. Schubert, S. Humphreys, C. Baroni, and M. Cohn, Proc. Nat. Acad. Sci. U.S. 64, 316 (1969). T. Amano, E. Richelson, and M. Nirenberg, Proc. Nat. Acad. Sci. U.S. 69, 258 (1972). e A. Blume, F. Gilbert, S. Wilson, J. Farber, R. Rosenberg, and M. Nirenberg, Proc. Nat. Acad. Sci. U.S. 67, 786 (1970). 7T. Amano, E. Richelson, and M. Nirenberg, Fed. Proc., Fed. Amer, Soc. Exp. Biol. 30, 1085 (1971). 8R. N. Rosenberg, L. Vandeventer, L. DeFrancesco, and M. E. Friedkin, Proc. Nat. Acad. Sci. U.S. 68, 1436 (1971). 9j. R. Kates, R. Winterton, and K. Schlessinger, Nature (London) 229, 345 (1971). lo D. Schubert, H. Tarikas, A. J. Harris, and S. Heinemann, Nature (London) N e w Biol. 233, 79 (1971). la N. W. Seeds, A. G. Gilman, T. Amano, and M. W. Nirenberg, Proc. Nat. Acad. Sci. U.S. 66, 160 (1970). 12D. Schubert, S. Hurnphreys, F. de Vitry, and F. Jacob, Develop. Biol. 25, 514 (1971). as j. Peacock, J. Minna, P. G. Nelson, and M. W. Nirenberg, Exp. Cell Res. 73, 367 (1972).

[77]

CULTURED CELL SYSTEMS, METHODS FOR NEUROBIOLOGY

769

T A B L E II GLIAL AND OTHER CLONAL CELL LINES

Cell type

Clone designation

Species

Glioma a,b,~

C6

Rat

Gliomab. ~

C2~

Rat

Gliomab,~ Glioma e Glioma ~

C2~ CHB l181N

Rat Uncertain Human

Glioma, transformed by polyoma virusl Peripheral neurinomaa Ependymoblastoma h Mast cell tumor ~,i

2 Five clones P-815-X-1

Rat Mouse Mouse

Mast cell tumor i,i

P-815-X-2

Mouse

Pituitary tumor a,k Pituitary tumor Z Pituitary tumor ~,k Melanoma, cloudman a,k Melanoma, cloudman a,'~ Melanoma, cloudman a,~ Melanoma ~ Melanomw~

AtT-20 GH-1 M-3 NCTC-3960 NCTC-3959 RPMI-1846 RPMI-3460-3

Mouse Rat Rat Mouse Mouse Mouse Syrian hamster Syrian hamster

Striated musclep

Many clones

Rat

Properties S100 protein; B-receptor; glial fibrils $100 protein; B-receptor; glial fibrils S100 protein S100 protein; B-receptor Histamine receptor; t~-receptor

Hamster S100 protein Tryptophan hydroxylase; histidine decarboxylase Tryptophan hydroxylase; histidine decarboxylase ACTH ACTH Growth hormone Melanin Melanin Amelanotic Melanin Melanin; 8-azaguanine resistant Contractile

a Available from the American Type Culture Collection, Rockville, Maryland. b p. Benda, J. Lightbody, G. Sato, L. Levine, and W. Sweet, Science 161,370 (1968). c p. Benda, K. Someda, J. Messer, and W. H. Sweet, J. Neurosurg. 34, 310 (1971). a j. Lightbody, S. E. Pfeiffer, P. L. Kornblith, and H. Herschman, J. Neurobiol. 1, 411 (1970). (Origin of cells is uncertain; personal communication, S. Pfeiffer.) e R. B. Clark and J. P. Perkins, Proc. Nat. Acad. Sci. U.S. 68, 2757 (1971). / H. M. Shein, J. Neuropathol. Exp. Neurol. 29, 70 (1970). g S. E. Pfeiffer, personal communication. h T. Gouaux, R. Ting, and M. Nirenberg, unpublished. R. Schindler, M. Day, and G. A. Fischer, Cancer Res. 19, 47 (1959). G. A. Fischer, Cancer Res. 19, 372 (1959). k y . Yasumura, A. It. Tashjian, and G. H. Sato, Science 154, 1186 (1966). G. H. Sato and Y. Yasumura, Trans. N.Y. Acad. Sci. 28, 1063 (1966). '~ K. K. Sanford, G. D. Likely, V. J. Evans, C. J. Mackey, and W. R. Earle, J. Nat. Cancer Inst. 12, 1057 (1966). G. E. Moore, D. F. Lehner, Y. Kikuchi, and L. A. Less, Science 137, 986 (1962). ° R. L. Davidson, B. Ephrussi, and K. Yamamoto, Proc. Nat. Acad. Sci. U.S. 56, 1437 (1966). C. Richler and D. Yaffe, Develop. Biol. 23, 1 (1970).

770

ISOLATION AND CULTURE OF CELLS

[77]

of adult mouse brain TM) with uncloned cells or with the neuroblastoma C-1300 tumor. Neuroblastoma and glioma clones have been hybridized with other cell types and with each other. 1~-17 Mutant parental cell lines that can be used to select hybrid progeny have been obtained. The properties of clonal gliomas and other cell types of neurobiological interest are given in Table II. In addition to clonal cell lines, striated muscle, ls-2° cardiac muscle, 21 and retinal pigment 22 cells can be cloned with high efficiency from embryonic tissue. Many studies with uncloned cells from the nervous system have been reported (for reviews, see references cited in footnotes 23 and 24). Many excellent articles on methods of cell culture are available, 25-~8 including those in previous volumes of "Methods in Enzymology." Such methods are not repeated here. Dissociation of Neural Tissue to Single Ceils Brain or other neural tissue is dissociated to single cells by incubation with trypsin or by extrusion of cells through nylon mesh. Reagents

Solution 1 : 1 3 7 mM NaC1; 5.4 mM KC1; 0.17 m M Na2HPO4; 0.22 m M KH_~PO4; 5.5 mM glucose; 3.4 /zM Phenol Red; and either 5.9 or 2.0 m M sucrose, respectively, when cells are to be grown with the Dulbecco-Vogt modification z9 of Eagle's medium, 340 mOsm/liter ( D M E M ) ; or Ham's F-12, 3° 300 mOsm/liter (Grand Island Biol. Co., Grand Island, New York) ,4 S. H. Wilson, J. L. Farber, R. Pertel, and M. Nirenberg, unpublished work. 1~R. L. Davidson and P. Benda, Proc. Nat. Acad. Sci. U.S. 67, 1870 (1970). l~j. Minna, D. Glazer, and M. Nirenberg, Nature (London) New Biol. 235, 225 (1972). 1, T. Amano, B. Hamprecht, W. Kemper, and M. Nirenberg, unpublished work. ~8I. R. Konigsberg, Develop. Biol. 26, 133 (1971). ~"N. K. White and S. D. Hauschka, Exp. Cell Res. 67, 479 (1971). "°C. Richler and D. Yaffe, Develop. Biol. 23, 1 (1970). -"~K. Goshima, Exp. Cell Res. 67, 352 (1971). 2~R. D. Cahn and M. B. Cahn, Proc. Nat. Acad. Sci. U.S. 55, 106 (1966). 2~M. R. Murray, hi "Cells and Tissues in Culture" (E. N. Willmer, ed.), Vol. 2, pp. 311 and 373. Academic Press, New York, 1965. 2, C. E. Lumsden, in "The Structure and Function of Nervous Tissue" (G. H. Bourne, ed.), Vol. I, p. 67. Academic Press, New York, 1968. ~ R. G. Ham and T. T. Puck, this series, Vol. 5, p. 90. L. Levintow and H. Eagle, this series, Vol. 5, p. 77. 2, R. D. Cahn, H. G. Coon, and M. B. Cahn, in "Methods in Developmental Biology" (F. H. Wilt and N. Wessells, eds.), p. 493. Crowell, New York, 1967. J. A. Boyle and J. E. Seegmiller, this series, Vol. 22, p. 149. 2, j. D. Smith, G. Freeman, M. Vogt, and R. Dulbecco, Virology 12, 185 (1960). ~R. G. Ham, Proc. Nat. Acad. Sci. U.S. 53, 288 (1965).

[77]

CULTURED CELL SYSTEMS, METHODS FOR NEUROBIOLOGY

771

Solution 2: 0.25% ( w / v ) crude trypsin (Nutritional Biochemicals Corporation, Cleveland, Ohio, 1:250), dissolved in solution 1, adjusted to pH 7.4 at 37 ° Solution 3: 90% solution 1 (or F-12 or D M E M ) and 10% fetal bovine serum Solution 4: 1% ( w / v ) nigrosin (Fisher Scientific Co., Pittsburgh, Pennsylvania), dissolved in solution 1 and adjusted to p H 7.4 Nitex nylon mesh cloth (Tobler, Ernst and Traber, Elmsford, New York) (about 12 × 12 cm) sterilized by autoclaving, 210 t~m pore diameter, and also 130 /~m pore diameter Trypsin Dissociation Procedure

Each step is performed under sterile conditions. Brain or other neural tissue in a known amount of solution 1 is weighed, washed with solution 1 at 3 °, and minced with scissors until pieces are less than 1 mm 3. The minced tissue is washed 3 times with solution 1, transferred with a wide-bore pipette to a flask containing solution 2 (100 ml per gram of tissue), and then incubated at 37 ° for 15 minutes with occasional swirling. Pieces of tissue are allowed to settle for 1 minute; the dissociated cells in the supernatant fraction are decanted and the suspension is immediately diluted with an equal volume of solution 3. Fresh solution 2 equal to the original volume is added to the undigested pieces of tissue and incubation and cell collection procedures are repeated. During incubation the pH is maintained at 7.4 by the addition of sterile 0.1 N NaOH. Five to 10 ml of solution 3 is added to the remaining undigested tissue, and the tissue is dissociated further by drawing the suspension into a 10-ml serological pipette and expelling the contents against the wall of the container (5 times). Dissociated cells then are recovered by decantation. Cells are pooled and sedimented at 250 g ...... for 10 minutes at 3 °, and the cells are suspended in solution 1. Cell concentration and viability are determined by mixing a small portion of the cell suspension with an equal volume of 1% nigrosin (solution 4) and counting cells within 10 minutes, using a hemacytometer. Cells that exclude the dye are assumed to be viable? l The relations between mouse brain wet weight and protein content as a function of age are shown in Fig. 1A. As shown in Fig. 1B, recovery of cells per mouse brain does not change with age, whereas the recovery of cells per gram of brain decreases markedly in older animals? 2 Recovery of cells, DNA, protein and enzyme activities are shown in Table III. Ap31j. p. Kaltenbach, M. H. Kaltenbach, and W. B. Lyons, Exp. Cell Res. 15, 112 (1958). ~2B. Schrier, R. Rosenberg, E. Thompson, and J. Farber, Fed. Proc., Fed. Amer. Soc. Exp. Biol. o.9, 480 (1970).

772

ISOLATION AND CULTURE OF CELLS A

i

i

[77]

i

B MOUSE BRAIN

• YIELD OF DISSOCIATED CELLS

o

400

EIGHT

300 Z Ilg I11

0

0

5C

200

R GRAM

I00

in C] Y

,

I

I0

i

I

20

i

I

I

30

o

,o

i

2'0

AOE OF

FIG. 1. Wet weight and protein content of mouse brains at various ages (panel A) and cell recovery by trypsin dissociation of various age mouse brains (panel B). Data are from B. Schrier, R. Rosenberg, E. Thompson, and J. Farber, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 9.9, 480 (1970).

proximately 15% of the cells from newborn mouse brain are recovered after dissociation, as estimated from the recovery of DNA in the cell fraction. Approximately 1 × l0 s cells are recovered per gram of newborn mouse brain tissue (95% of the cells are viable). The specific activities of choline acetyltransferase, glutamate decarboxylase, and acetylcholinesterase after dissociation of mouse brain tissue are 36, 24, and 52%, respectively, of values found prior to dissociation. 1 S i e v i n g D i s s o c i a t i o n P r o c e d u r e ~3-~

Brain tissue is weighed and washed as described above. Tissue is placed in the center of a 12 × 12 cm piece of nylon cloth (210 ~m pore diameter), the ends of the nylon screen are gathered with forceps, and the tissue, now enclosed in one layer of nylon cloth, is submerged in solution 1 at 24 ° (20 ml per gram of tissue). Cells then are extruded by squeezing the bag against the surface of the container with a glass rod. The resulting suspension of cells and tissue is filtered through a single thickness of nylon cloth (130-~m pore diameter). The filtrate containing dissociated cells is CO1aaS. P. R. Rose, Nature (London) 206, 621 (1965). a'S. Varon and C. W. Raiborn, Jr., Brain Res. 12, 180 (1969). 3~B. K. Schrier, 1. Neurobiol. 4, 117 (1973).

[77]

CULTURED

CELL

x

SYSTEMS,

METHODS

FOR

NEUROBIOLOGY

773

X

R

Z © 0

i

0

O0

cO

o z

N QO

N

z

N

©

•~

0 0

o

~o

-~ ~ , ' ~ -~ o o ~ ~ o

X

774

ISOLATION AND CULTURE OF CELLS

[77]

lected in a centrifuge tube. Approximately 3.5 × 108 cells (50% viable) are recovered per gram of brain from 20-day rat fetuses. Enzyme specific activities of dissociated cells from mouse or rat brain cultured for various times either in monolayerT M or shaker culture 37 are shown for comparative purposes in Table III. Preparation of Homogenates The procedure is designed to wash cell monolayers free of serum protein and to recover enzyme activities reproducibly and in high yield.

Reagents and Apparatus Solution 1 : 1 3 7 mM NaC1; 5.4 mM KC1; 0.17 mM Na2HPO,; 0.22 mM KH~PO4; 5.5 mM glucose; 0.14 mM CaCI~ and 5.9 mM sucrose, adjusted to pH 7.4 with 0.1 N NaOH (340 mOsm/liter) Solution 2: Same as solution 1 except without CaCI~ Solution 3 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt Solution 4 : 1 0 0 mM potassium phosphate buffer, pH 6.2 Spatula: End of a flexible plastic ruler, 5 cm in width, covered with disposable Teflon tape (Scientific Specialties Services, Inc., Randallstown, Maryland) Sonic oscillator (Raytheon Co., Arlington, Virginia, Model No. DF101)

Procedure. Growth medium is removed from a petri dish (150 mm in diameter), and the cell monolayer is gently washed twice with 10-ml portions of solution 1 and once with 10 ml of solution 2. The petri dish is inverted and drained at a 315 o angle for 90 seconds, then the dish is scraped with a Teflon-covered spatula. Cells are recovered by aspiration with a large-bore micropipette and transferred to a 3 × 0.5 inch polyallomer tube in an ice-water bath. The surface of the dish is washed twice with 0.1-1.5 ml portions of solution 3 (all assays except tyrosine hydroxylase) or solution 4 (tyrosine hydroxylase assay) at 3 °. The volume of solution 3 or 4 added is adjusted so that the final protein concentration is 2-10 mg/ml of homogenate. Cells can be recovered from the washes by centrifugation and combined with the scraped ceils. Each tube is stoppered, and placed in cold H20 in the chamber of the sonic oscillator; cells then are lysed by sonication at 1 ° for 5 minutes. Homogenate volumes are measured; each homogenate is divided into small portions which are frozen quickly and stored in the vapor phase of a liquid nitrogen freezer. 3"D. L. Shapiro and B. K. Schrier, Exp. Cell Res. 77, 289 (1973). SrN. W. Seeds, Proc. Nat. ,4cad. Sci. U.S. 68, 1959 (1971).

[77]

CULTURED CELL SYSTEMS, METHODS FOR NEUROBIOLOGY

775

Reaction mixtures are prepared at 1 °; homogenates are added to reactions last. Enzyme activity is determined at 4 concentrations of homogenate protein. Portions of homogenates are assayed for protein by the method of Lowry et alY 8 and for DNA by the method of Kissane and Robins. 39 Acetylcholinesterase (EC 3.1.1.7) Assay [1-'4C]Acetylcholine -I- H20 ~ [1-uC]aeetate q- choline Principle. A modification 1,6 of the assays of Reed et al. 4° and Ehrenpreis et al. ~1 is described. [1-14C]Acetylcholine is hydrolyzed in the presence of

enzyme extract; the reaction mixture is passed through a column of a cation-exchange resin, which retains the radioactive substrate but not the [14C]acetate product. Radioactivity in the column effluent is determined using a liquid scintillation counter. Reagents and Apparatus

Solution 1 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 1.0M NaC1; and 2.5% (v/v) Triton X- 100 (Packard Instrument Co., Downers Grove, Illinois) Solution 2 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 0.2 M NaC1; and 0.5% Triton X-100 Solution 3 (reaction cocktail): 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 0.5% Triton X-100; and 14 mM [1-14C]acetylcholine iodide (0.35 mCi/mmole; 0.05 /~Ci/ reaction mixture). Before preparing solution 3, radioactive acetylcholine preparations are dissolved in HzO and lyophilized for 16 hours to remove possible volatile contaminants. Cation exchange resin: AG 50W-X8 (H + form, 100-200 mesh, BioRad Laboratories, Richmond, California) is incubated with 2 N NaOH at 25 o for 45 minutes, washed with H20 until the effluent pH is 6.0, and then stored at 4 °. Columns (0.5 × 5 cm) of resin are formed over small plugs of glass wool in 9-inch disposable Pasteur pipettes, one column for each reaction, and washed with H20. Solution 4 : 2 0 0 0 ml of toluene, 1000 g of Triton X-100, and 165 ml of Liquifluor (New England Nuclear, Boston, Massachusetts) Solution 5 : 9 6 8 ml of toluene and 42 ml of Liquifluor ~8O. H. Lowry, N. F. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 39j. M. Kissane and E. Robins, J. Biol. Chem. 233, 184 (1958). ~°D. J. Reed, K. Goto, and C. H. Wang, Anal. Biochem. 16, 59 (1966). 41S. Ehrenpreis, A. Chiesa, M. Bigo-Gullino, and P. Patrick, Fed. Proc., Fed. Amer. Soc. E x p . B i o l . 26, 296 (1967).

776

ISOLATION AND CULTURE OF CELLS

[77]

Procedure. Four volumes of homogenate are mixed with 1 volume of solution 1 just prior to assay. Each 50-~1 reaction contains 10 ~I of solution 3 and 0-40 ~1 of homogenate and/or solution 2. The final concentration of each component is: 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 2.80 mM [1-14C]acetylcholine iodide (0.05 ~Ci/reaction mixture, 0.35 mCi/mmole), 200 mM NaC1, and 0.5 % Triton X-100. Reactions in 10 × 75 mm disposable glass tubes are incubated for 10 minutes at 37 °, then transferred to an ice-water bath and diluted rapidly by the addition of 1.0 ml of H_~O at 1 °. Each diluted reaction is immediately passed over a column of the cation exchange resin; the tube is washed with two 1.0-ml portions of H,~O at 1 °, and the washes also are passed through the column. The column effluent is collected in a glass scintillation vial, then 10 ml of solution 4 are added, and radioactivity is determined with a liquid scintillation counter. Since reactions are not deproteinized, the column step must be performed rapidly. The rate of acetylcholinesterase activity after dilution at 1 ° is approximately 1.5% that of undiluted reactions at 37 °. The assay is performed in batches of 20 tubes or less so that all reactions can be passed through columns within 3 minutes. lnhibitors. A number of enzymes catalyze acetylcholine hydrolysis, including ACHE, cholinesterase (EC 3.1.1.8), and acetylesterase (EC 3.1.1.6). The enzymes differ from one another in relative activity toward various substrates and in sensitivity to inhibitors. Diisopropyl fluorophosphate, an inhibitor of ACHE, cholinesterase, and carboxylesterase (EC 3.1.1.1), but not arylesterase (EC 3.1.1.2), resulted in a 50% inhibition of neuroblastoma C-1300 AChE at a concentration of 10 nM2 The activity was also inhibited ~ 50% by 60 nM 1,5-bis-4-allyldimethylammoniumphenylpentane-l,3-dibromide (BW 284C51), a more potent inhibitor of AChE than cholinesterase42; by 0.7 ~M neostigmine sulfate, a more potent inhibitor of AChE and cholinesterase than arylesterase or carboxylesterase, and by 0.4 mM tetramonoisopropylpyrophosphortetramide, a more potent inhibitor of cholinesterase than ACHE. '~ Product Identification. Reaction mixtures are prepared and incubated as described above. Just prior to the ion-exchange chromatography step, sodium [3H]acetate is added to the reaction mixture to adjust the ratio of 3H:14C dpm expected in the column effluent to 4:1. Sodium hydroxide is added to a portion of the column effluent so that the final concentration is 0.18 N. This solution and a portion of the effluent without sodium hydroxide are spotted on Whatman paper or cellulose-coated thin-layer sheets for ascending chromatography in the following solvent systems:

'2L. Austin and W. K. Berry, Biochem. J. 54, 695 (1953).

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

777

Solvent I1: Ethanol:conc. NH4OH:H20 = 8:1:1 (R I of acetate = 0.70) Solvent II 1: 1-butanol:cyclohexane: ethylene glycol:H.~O: conc. NH,OH:cyclohexylamine = 30: 30:10:3.7:0.07:0.05 (RI of acetate = 0.25) Solvent IIP (to detect alkali-labile radioactive contaminants): 1-propanol:0.1 N acetic acid = 3:1. Chromatography should be performed in a fume hood since the radioactive acetate is volatile in this solvent system. Developed and dried chromatograms, unstained, are cut into strips and placed in scintillation vials containing solution 5; radioactivity is determined with a liquid scintillation counter. Catechol O-Methyltransferase (EC 2.1.1.1) Assay S-Adenosyl-[methylA4C]methionine-1- 3,4-dihydroxybenzoicacid 3-[14C]methoxy-4-hydroxybenzoicacid q4-[14C]methoxy-3-hydroxybenzoicacid q- S-adenosylhomocysteine Principle. Neuroblastoma homogenates are assayed for catechol O-methyltransferase activity by a modificationl of the method of Nikodejevic et al. 4:~ The [1~C] reaction products are separated from the labeled substrate by extraction with toluene, and the radioactivity of the toluene layer then is determined. R e a g e n t s and A p p a r a t u s

Solution 1 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; and 50 mM MgCI~ Solution 2 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; and 5 mM MgC12 Solution 3 (reaction cocktail): 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt: 5 mM MgC12; 12.5 mM 3,4-dihydroxybenzoic acid; and 2.75 mM L-S-adenosyl-[methyl14C] methionine iodide (1.55 mCi/mmole, 0.32 t~Ci/reaction mixture). The radioactive S-adenosylmethionine is extracted twice with 10 ml of toluene and the aqueous phase is lyophilized before use. Solution 4 (for terminating reactions): 1.0 N hydrochloric acid Reagent grade toluene

43B. Nikodejevic, S. Senoh, J. W. Daly, and C. R. Creveling, 1. Pharmacol. Exp. Ther. 174, 83 (1970).

778

ISOLATION AND CULTURE OF CELLS

[77]

Solution 5:958 ml of toluene and 42 ml of Liquifluor (New England Nuclear) Conical centrifuge tubes, 12 ml, with ground glass stoppers

Procedure. Homogenates are thawed immediately before use and 1 volume of solution 1 is added to 9 volumes of homogenate. Each reaction contains 10/~1 of solution 3, 0-40 ,,~1of neuroblastoma homogenate (added last), and solution 2 as needed to adjust the final volume to 50 /zl. The final concentration of each reaction component is as follows: 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 0.55 mM L-S-adenosyl-[methyl-~4C]methionine iodide (0.32 /~Ci/reaction mixture, 11.55 mCi/mmole); 2.5 mM dihydroxybenzoic acid; 5 mM MgC12; and 0-500/zg of neuroblastoma homogenate protein. Reaction mixtures in 12 ml conical glass centrifuge tubes are incubated for 20 minutes at 37 °, transferred to an ice-water bath, and 0.2 ml of solution 4 and 10 ml of toluene are added. Stoppered tubes are shaken vigorously for 1 minute and centrifuged for 5 minutes at 250 g at 3 °. Nine milliliters of the toluene phase are transferred to a scintillation vial containing 5 ml of solution 5, and radioactivity is determined with a liquid scintillation counter. A correction is applied so that each value corresponds to the entire toluene phase. lnhibitors. Neuroblastoma clone N-18 catechol O-methyltransferase activity is inhibited approximately 80% by 0.25 mM 3,5-dihydroxy-4methoxybenzoic acid (Regis Chemical Co., Chicago, Illinois) or by 1 mM tropolone (Aldrich Chemical Co., Cedar Knolls, New Jersey). In the absence of these compounds approximately 70 and 5 pmoles of 3-methoxy-4hydroxybenzoic acid and 4-methoxy-3-hydroxybenzoic acid, respectively, are formed per minute per milligram of neuroblastoma clone N-18 protein) Product Identification. The reaction volume may be increased 5-fold, if desired. Reactions are extracted with toluene, and the toluene phase is evaporated to dryness under a stream of nitrogen. The residue is dissolved in a small volume of methanol. Authentic nonradioactive (A) 3-methoxy-4hydroxybenzoic acid (Sigma Chem. Co., St. Louis, Missouri) and (B) 4-methoxy-3-hydroxybenzoic acid (K & K Laboratories, Inc., Plainview, New York) are added, and portions of the extract containing 50 t~g of each are spotted on thin-layer plates of silica gel G (Eastman Organic Chemicals, Rochester, New York) and developed in the following solvent systems: Solvent I: 2-propanol:conc. NH4OH:H20 = 8:1:1 (R: of A = 0.38, R: of B = 0.76) Solvent II: 1-butanol : formic acid: benzene: H20 = 30: 50: 10: 3 (R: of A = 0.91, R: of B = 0.75) Solvent III: Benzene : methanol: glacial acetic acid = 10: 5 : 1 (R: of A = 0.86; R: of B = 0.75)

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

779

Chromatograms are dried, and catechols are located under UV light. Then chromatograms are cut into strips, placed in scintillation vials, covered with solution 6, and radioactivity is determined with a liquid scintillation spectrometer. Glutamate Decarboxylase (EC 4.1.1.15) Assay [U-l~C]Glutamic acid --)

[14C]02

"~ 3,-amino-[UJ4C]bul.yricacid

Principle. A modification' of the assay of Wingo and Awapara 44 is described. The 14CO~ formed is absorbed by a solution of Hyamine hydroxide, and the radioactivity then is determined. Since 14CO_, also can be generated from glutamic acid via other pathways, the 7-amino-[14C]butyric acid product of the reaction also is identified by paper electrophoresis and thin-layer chromatography, and the amounts of 7-amino-[14C]butyric acid and 14CO_, formed are compared. Reagents and Apparatus

Solution 1 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 10 mM 2-mercaptoethanol; 5% (v/v) Triton X-100 (Packard Instrument Co., Downers Grove, Illinois), and 5 mM pyridoxal phosphate Solution 2 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 1 mM 2-mercaptoethanol: 0.5% Triton X-100; and 0.5 mM pyridoxal phosphate Solution 3 (reaction cocktail): 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 1 mM 2-mercaptoethanol; 0.5% Triton X-100; 0.5 mM pyridoxal phosphate; and 25 mM L-[U-14C]glutamic acid, potassium salt (4.16 mCi/mmole, 1.04 Ci/reaction mixture). The radioactive glutamic acid is neutralized with KOH and lyophilized before use. Solution 4 : 6 4 0 ml of toluene; 288 ml of 1 M Hyamine hydroxide in methanol (Packard Instrument Co.); 42 ml of Liquifluor (New England Nuclear) Solution 5 (for terminating reactions): 10 mM acetic acid in methanol Disposable glass tubes, 10 × 37 mm Rubber stoppers with flanges (A. H. Thomas, Philadelphia, Pennsylvania), Cat. No. 1781-E90). The stoppers fit the 10 × 37 mm test tubes; the flanges fit scintillation vials with mouths of 16-ram internal diameter. "W. J. Wingo and J. Awapara, J. Biol. Chem. 187, 267 (1950).

780

ISOLATION AND CULTURE OF CELLS

[77]

Scintillation vials with mouths of 16 mm diameter (internal diameter) Solution 6 : 9 5 8 ml of toluene and 42 ml of Liquifluor (New England Nuclear)

Procedure. Homogenates are thawed prior to use and adjusted to the conditions of the glutamate decarboxylase assay by mixing 9 volumes of homogenate with 1 volume of solution 1. Each reaction contains the following components in a final volume of 50 ~1:50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 5 mM L-[U-l'C]glutamic acid, potassium salt (4.16 mCi/mmole, 1.04 ~Ci/reaction mixture); 1 mM 2-mercaptoethanol; 0.5 mM pyridoxal phosphate; 0.5 % Triton X-100; and 0--500/~g of mouse brain homogenate protein. Reaction mixtures are prepared in 10 × 37 mm glass test tubes, each tube containing 10 ~1 solution 3, 0-40/zl of homogenate (added last), and solution 2 as needed to adjust the final volume to 50 ~1. The test tube then is sealed with a tightly fitting flanged rubber stopper. Reactions are incubated for 10 minutes at 37 ° and then transferred to an ice-water bath. Each tube is placed in a glass scintillation vial containing 5 ml of solution 4. The scintillation vial is sealed with the flange of the rubber stopper which remains on the 10 x 37 mm test tube containing the reaction mixture. Then 0.2 ml of solution 5 is injected with a hypodermic syringe through the rubber stopper into the reaction mixture in the test tube. The 10 X 37 mm test tube is dislodged from the rubber stopper with the injection needle so that the test tube falls to the bottom of the scintillation vial. The sealed scintillation vial is incubated for 30 minutes at 24 ° to allow absorption of ~4CO~ by the Hyamine solution. Then the rubber cap is removed from the scintillation vial, the reaction tube is removed from the scintillation vial with forceps, and the scintillation vial is capped with a scintillation vial top. Radioactivity is determined with a liquid scintillation counter (80% counting efficiency for ~C). lnhibitors. Hydroxylamine and related compounds have been shown to inhibit 14CO2 production from [1-1'C]glutamate. 45 Identification of y-Amino-[~'C]butyric AcM. The reaction volume may be increased to 0.25 ml without altering the concentrations of the components described above. After the reaction is incubated, 0.2 ml of 10 mM acetic acid in methanol is added, and COo is absorbed in the usual manner; the reaction then is centrifuged at 15,000 g for 10 minutes at 3 °. The supernatant fluid is removed and subjected to paper electrophoresis and thin-layer chromatography (MN Polygram Cel 300, Brinkman Instruments, Inc., Westbury, New York) with the following solvent systems: 45E. Roberts and D. G. Simonsen, Biochem. Pharmacol. 12, 113 (1963).

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

781

Paper Electrophoresis Solvent I: Pyridine:glacial acetic acid:water = 36:100:2890, pH 3.95, 45 minutes at 61 V/cm and 180 mA

Thin-Layer Chromatography Solvent II: 2-propanol:methanol:conc. NH4OH = 9:7:4 (R I of 7-aminobutyric acid is 0.42) Solvent III: Phenol saturated with 6 M NH4OH (R~ of ,/-aminobutyric acid is 0.88) Solvents IV and V: Chromatography in 2 dimensions: Solvent IV, first dimension: 2-propanol:l-butanol:l N HC1 = 12 : 3 : 5 (Rs of ],-aminobutyric acid is 0.77) Solvent V, second dimension: Phenol saturated with H~O (Ry of 7-aminobutyric acid is 0.89) Chromatograms and electropherograms are dried, stained with 0.2% ninhydrin in acetone, and cut into small strips; each strip is placed in a scintillation vial. Ten milliliters of solution 6 is added to each vial, and radioactivity is determined with a liquid scintillation counter. Choline Acetyltransferase (EC 2.3.1.6) Assay I [1-14C]Acetyl-CoA+ choline~- [1-14C]acetylcholine+ CoASH Two methods of assay modified from the assay reported by Schrier and Shuster ~6 are described in this section and the one following. In the method described in this section 1 the incubated reaction mixture is passed through a disposable column of anion exchange resin under conditions where the radioactive substrate is retained and radioactive products are recovered in the column effluent. Chromatographic identification of products is necessary because radioactive acetylcarnitine and other products may be present in the column effluent in addition to acetylcholine? An alternative method of assay, described in the following section, employs acetylcholinesterase to characterize the product.

Reagents Solution 1 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 1.0 M NaC1; and 2.5% (v/v) Triton X-100 (Packard Instrument Co., Downers Grove, Illinois) Solution 2 : 5 0 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA, potassium salt; 0.2 M NaC1; and 0.5% Triton X-100 Solution 3 (reaction cocktail): 50 mM potassium phosphate buffer, ~"B. K. Schrier and L. Shuster, J. Neurochem. 14, 977 (1967).

782

ISOLATION AND CULTURE OF CELLS

[77]

pH 6.8; 1 mM EDTA, potassium salt; 0.5% Triton X-100; 0.5 mM neostigmine methylsulfate; 10 mM choline chloride; and 1.1 mM [l-14C]acetyI-CoA (6 mCi/mmole, 0.05 /~Ci/reaction). The radioactive acetyl-CoA is lyophilized before use. Anion exchange resin: AG I-X8 (chloride form, 100--200 mesh, Bio-Rad Laboratories, Richmond, California) is washed with H20 until the effluent is pH 5.5-6.0. Columns (0.5 × 5 cm) of resin are formed over small plugs of glass wool in 9-inch disposable Pasteur pipettes and washed with H_oO. Solution 4:2000 ml of toluene; 1000 g of Triton X-100, and 165 ml of Liquifiuor (New England Nuclear) Solution 5:958 ml of toluene and 42 ml of Liquifluor Procedure. Homogenates are thawed prior to use and adjusted to the conditions of the choline acetyltransferase assay by mixing 4 volumes of homogenate with 1 volume of solution 1. Each 50 ~tl reaction contains 10 /~1 of solution 3 and 0-40 ~1 of homogenate and/or solution 2. The final concentration of each component is: 50 mM potassium phosphate buffer, pH 6.8; 1 mM EDTA potassium salt; 0.21 mM [1-14C]acetyl-CoA (6 mCi/mmole, 0.05 #Ci/reaction mixture); 2 mM choline chloride; 200 mM NaCI; 0.1 mM neostigmine methylsulfate and 0.5% Triton X-100; and 0-0.75 mg of homogenate protein. The rate of reaction is proportional to protein concentration within the range of 10-300 ~,g of protein from neuroblastoma homogenates. Reactions are incubated in 10 × 75 mm glass tubes for 10 minutes at 37 ° , then transferred to an ice-water bath and diluted by the addition of 1.0 ml of H~O at 1°. The contents of each tube are passed through an anion exchange column; the tube is washed with two 1.0-ml portions of H20 at 1°, and the washes are also passed through the column. The column effluent is collected in a glass scintillation vial, then 10 ml of solution 4 are added and radioactivity is determined with a liquid scintillation counter. Inhibitors. Several styryl-pyridine analogs have been characterized as inhibitors of choline acetyltransferase.'~,4s Product Identification. Reaction mixtures are prepared and incubated as described above. The chloride salts of both [:~H]acetylcholine and choline should be used to avoid complications in product identification by chromatography. Just prior to the ion-exchange chromatography step, [3H]acetylcholine chloride is added to the reaction mixture to adjust the ~H: 1"C dprn ratio expected in the column effluent to 4: 1. Glacial acetic acid is added to the column effluent so that the final concentration is

47j. C. Smith, C. J. Cavallito, and F. F. Foldes, Biochem. Pharmacol. 16, 2438 (1967). 'SH. L. White and C. J. Cavallito, Biochim. Biophys. Acta 206, 242 and 343 (1970).

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

783

0.06 M. Portions of the effluent are then spotted on Whatman paper or cellulose-coated thin-layer sheets (MN Polygram Cel 300, Brinkman Instruments, Inc., Westbury, New York) for ascending chromatography, and developed as follows: Solvent 149: 1 - p r o p a n o l : 0 . 1 N acetic acid = 3:1 ( R I of 0.66) Solvent 1149: 1-propanol:benzyl alcohol:H20 = 5 : 2 : 2 ACh = 0.89 and 0.65) Solvent III4'~: 1-propanol:formic acid:H20 = 8 : 1 : 1 (Rs of 0.77) Solvent IV4~: 1-butanol: 1-propanol:H20 = 4 : 2 : 1 (Rs of 0.49)

ACh = (RI

of

ACh = ACh =

Developed and dried chromatograms are cut into strips (without staining), placed in scintillation vials and covered with solution 5, and radioactivity is determined with a liquid scintillation spectrometer adjusted for double-label counting. Characterization of acetylcholine by paper electrophoresis has been described by Potter and colleagues, t° ~3 Choline Acetyltransferase (EC 2.3.1.6) Assay II B y BERND HAMPRECHT, TAKEHIKO AMANO, and MARSHALL NIRENBERG

[1-14C]Acetyl-CoAq- choline ,~- [1J4C]acetylcholine -b CoASH [1-14C]Aeetylcholineq- H20 ---*[1J4C]acetate -b choline

(1) (2)

In assay I, described in the preceding section, [14C]acetylcholine formed in reaction 1 is separated from other radioactive components by passing the mixture through an anion exchange column and subjecting the effluent to paper electrophoresis or chromatography. Alternatively, the amount of acetylcholine formed can be determined by the following differential method~4: For each type a reaction mixture (assay I slightly modified), a second reaction mixture (type b, with acetylcholinesterase instead of neostigmine) is prepared. In type b reactions the acetylcholine formed is hydrolyzed with high specificity, leaving contaminants, such as acetylcarnitine, unchanged. On subsequent passage through an anion exchange column the eluate contains radioactive contaminants, but not acetylcholine. 4.~V. P. Whittaker, in "Handbuch der Experimentellen Pharmakologie" (O. Eichler and A. Farah, eds.), p. 2. Springer-Verlag, Berlin and New York, 1963. ~°L. T. Potter, V. A. S. Glover, and J. K. Saelens, J. Biol. Chem. 243, 3864 (1968). ~1L. T. Potter and V. A. S. Glover, this series, Vol. 17B, p. 798. 52V. A. S. Glover and L. T. Potter, J. Neurochem. 18, 571 (1971). ~3L. T. Potter and W. Murphy, Biochem. Pharmacol. 16, 1386 (1967). ~4B. Hamprecht and T. Amano, Analyt. Biochem. 57, 162 (1974).

784

ISOLATION AND CULTURE OF CELLS

[77]

The amount of acetylcholine formed is determined from the difference between the radioactivity values obtained from the parallel reaction mixtures a and b.

Reagents Solution 1 : 5 0 mM potassium phosphate buffer, pH 6.8; 0.2M NaC1; 0.5% Triton X-100 (Packard Instrument Co., Downers Grove, Illinois) Solution 2: Neostigmine methylsulfate, 1 mM, in solution 1 Solution 3: Acetyl-CoA, 5 mM in solution 1; stored frozen. The concentration of the solution is determined spectrophotometrically.55 Solution 4: Choline iodide, 6.25 x 10 -2 M in solution 1 [1-1'C]Acetyl-CoA: 1 ~mole of radioactive acetyl-CoA (50 mCi/ mmole, New England Nuclear), dissolved in 2.5 ml of HzO, is purified as follows: The solution is applied to a 0.5 x 3 cm column of Bio-Rad 50W-X8, 100-200 mesh, Na + form, in a 9-inch Pasteur pipette with glass wool plug. The column is washed with 2 ml of H20 and the radioactivity of the pooled eluates is determined. The pooled eluates are lyophilized. Solution 5 (reaction cocktail, 10x ) : The purified [1-14C]acetyl-CoA is dissolved in adequate volumes of solutions 1, 3, and 4 to yield a cocktail containing 50 mM potassium phosphate buffer, pH 6.8; 200 mM NaC1; 0.5% Triton X-100; 12.5 mM choline iodide; 2.5 mM [1-14C]acetyl-CoA (15-30 mCi/mmole; according to the required sensitivity of the assay). The radioactivity of the cocktail and its acetyl-CoA content 5~ are determined to evaluate the specific radioactivity. Solution 6 : 3 mg of acetylcholinesterase protein (5000 units) (EC 3.1.1.7) from Electrophorus electricus (Worthington Biochemical Corporation, Freehold, New Jersey), is dissolved in 2.5 ml of solution 1, divided into small portions, and stored in the vapor phase of a liquid nitrogen freezer. Anion exchange resin: AG1-X8 (C1- form, 100-200 mesh, BioRad) is washed with water until the effluent pH is 5.5-6.0. Columns of resin (0.5 x 5.0 cm) are formed over glass wool plugs in 9-inch Pasteur pipettes and washed with water. Solution 7:2000 ml of toluene, 1000 g of Triton X-100, and 165 ml of Liquifluor (New England Nuclear) Cell homogenates are prepared as described above, using solution 1. 5°W. Buckel and H. Eggerer, Biochem. Z. 343, 29 (1965).

[77]

CULTUREDCELL SYSTEMS, METHODS FOR NEUROBIOLOGY

785

P r o c e d u r e . Homogenates are thawed immediately before use. Each type a reaction mixture with neostigmine contains the following components in a final volume of 50 ~1:50 mM potassium phosphate buffer, pH 6.8; 200 mM NaCI; 0.5% Triton X-100; 0.25 mM [1-~4C]acetyl-CoA ( 1 5 - 3 0 m C i / m m o l e ) ; 1.25 mM choline iodide; 0.1 mM neostigmine methylsulfate; 40 ~1 of homogenate a n d / o r solution 1. Each type b reaction contains the same components as type a except that 10 units of acetylcholinesterase is added in place of neostigmine methylsulfate. The absence of E D T A did not affect the rates of reactions 1 and 2. Each reaction mixture in a 10 × 75 mm glass tube is incubated for 10 minutes at 37 °, then the tube is transferred to an ice-water bath and 1.0 ml of ice-cold H,.,O is added. The diluted reaction mixture is passed through a column of anion exchange resin; the tube is washed with two 1.0 ml portions of ice-cold H20, and the washes also are passed through the column. The column effluent is collected in a scintillation vial, then 10 ml of solution 7 are added and radioactivity is determined with a liquid scintillation counter. The recovery of acetylcholine is 98% when the reaction mixture obtained in the presence of neostigmine is passed over the column. Each homogenate is assayed at 4 concentrations in the presence of neostigmine as well as in the presence of acetylcholinesterase. Blank values are obtained under both types of conditions in the absence of homogenate. They are subtracted from the corresponding values obtained with homogenate. From the values thus found for the reaction in the presence of neostigmine, those found for the reaction in the presence of acetylcholinesterase are subtracted. P r o d u c t Identification. Methods for the identification of acetylcholine are described in the preceding section.

Tyrosine Hydroxylase (EC 1.14.3a) Assay B y ELLIOTT RICHELSON and MARSHALL NIRENBERG

L-[3,5-3H]Tyrosine+ 02 + tetrahydropteridine [3H]OH + L-3,4-dihydroxy-[5-~H]phenylalanine-I- dihydropteridine Principle. A modification of the assays of Shiman and Kaufman, 56 Shiman et al., 57 and Nagatsu et al. 5s is described. Formation of dihydroxyphenylalanine results in the release of ~H÷ from the 3-position of L-[3,5-~H] tyrosine. The reaction mixture is passed through a column of cation exchange resin, resulting in the retention of [:~H]amino acids but not of 3HOH.

R. Shiman and S. Kaufman, this series, Vol. 17A, p. 609. *'R. Shiman, M. Akino, and S. Kaufman, J. Biol. Chem. 246, 1330 (1971). ~ST. Nagatsu, M. Levitt, and S. Udenfriend, J. Biol. Chem. 239, 2910 (1964).

786

ISOLATION AND CULTURE OF CELLS

[77]

The radioactivity of the ~HOH in the column effluent is determined with a liquid scintillation counter.

Reagents Solution 1 : 1 0 0 mM potassium phosphate buffer, pH 6.2 Solution 2 (reaction cocktail) : 250 mM potassium phosphate buffer, pH 6.2; 0.625 mM NADPH, tetrasodium salt; 0.75 mM 2-amino4-hydroxy-6,7-dimethyltetrahydropteridine (Calbiochem, La Jolla, California); 5000 units of catalase (crystalline, approximately 87,000 units/mg protein, Worthington Biochemical Corporation, Freehold, New Jersey); 1.38 mg of sheep liver pteridine reductase protein per milliliter (purified through the second ammonium sulfate step according to KaufmanS'Q; and 1.25 mM L - [ 3 , 5 - 3 H ] tyrosine (Amersham/Searle Corporation, Des Plaines, Illinois), 13.6 mCi/mmole, 0.34 ffCi/reaction. Before preparing solution 2 the tritiated tyrosine (1000 mCi/ mmole) is purified in the following manner: 0.15 ttmoles of tritiated tyrosine dissolved in 150 ffl of 2% aqueous ethanol is adjusted to 0.1 N acetic acid by the addition of glacial acetic acid and then adsorbed onto a 0.5 x 5 cm column of cation exchange resin (described below) and the column then is washed with 300 ml of H20. The tritiated tyrosine is eluted with 15 ml of 3 N HCI; fractions containing the peak of radioactivity are pooled and evaporated to dryness with a flash evaporator. The tritiated tyrosine is dissolved in approximately 2 ml of 2% ethanol, divided into portions, and then stored in the vapor phase of a liquid nitrogen freezer for < 3 weeks. Immediately before use the solution is thawed, lyophilized and dissolved in solution 2 that lacks tritiated tyrosine. Solution 3 : 0 . 1 7 N acetic acid at 4 ° Cation exchange resin: AG 50W-X8 (H ÷ form, 100-200 mesh, BioRad) is washed in succession with 2 N NaOH, 4 N HCI, and then with H~O until the pH of the effluent is 6; and stored at 4 °. Columns (0.5 X 5 cm) of resin are formed over small plugs of glass wool in 9-inch disposable Pasteur pipettes, one column for each reaction, and washed with H20. Solution 4:2000 ml of toluene, 1000 g of Triton X-100, and 165 ml of Liquifiuor (New England Nuclear) Solution 5 (used for product identification): Solution 2 with 0.25 mM p-bromo-rn-hydroxybenzyloxyamine 5.~S. Kaufman, this series, Vol. 5, p. 812.

[77]

CULTURED CELL SYSTEMS, METHODS FOR NEUROBIOLOGY

787

Alumina gel: 200 mg of aluminum oxide (British Drug Houses, Ltd., Poole, Dorset, England) prepared according to Udenfriend,G° is added to a beaker containing 10 ml of 200 mM sodium acetate and 0.5 ml of 200 mM EDTA, sodium salt. Solution 6 : 0 . 2 N HC1 Solution 7 (spray reagent for chromatographyG1) : 0.1 g of potassium ferricyanide dissolved in 45 ml of absolute ethanol, 5 ml of ethylenediamine (Eastman Organic Chemicals, Rochester, New York) and 50 ml of H,_,O Solution 8:0.01 N HCI Solution 9 : 5 mM L-3,5-dihydroxyphenylalanine (Calbiochem) Solution 10: 5% (w/v) trichloroacetic acid at 3 ° P r o c e d u r e . Homogenates are prepared as described above except that 100 mM phosphate buffer, pH 6.2, is used. Each reaction contains the following components in a final volume of 50 tzl: 160 mM potassium phosphate buffer, pH 6.2; 0.5 mM L-[3,5-~H]tyrosine (13.6 mCi/mmole, 0.34 t~Ci/reaction mixture); 0.25 mM NADPH; 0.3 mM 2-amino-4-hydroxy6,7-dimethyltetrahydropteridine; 40 units of catalase (0.45 /~g protein); 28 ~g of pteridine reductase protein; and 0-500 t~g of neuroblastoma homogenate protein. Each reaction in a 10 X 75 mm glass test tube contains 20/~1 of solution 2; 0-30/~1 of homogenate (added last) and enough solution 1 to adjust the final volume to 50/zl. Tubes are incubated for 10 minutes at 34°; each reaction is terminated by the addition of 0.5 ml of solution 3, then the suspension is passed through a column of cation exchange resin. The tube is washed with two 0.75-ml portions of H20 at 3 ° and the washes also are passed through the column. The column effluent is collected in a glass scintillation vial; 10 ml of solution 4 is added, and radioactivity is determined with a liquid scintillation counter. At 30% counting efficiency, the usual value in the absence of enzyme is 350 cpm (range, 150-800 cpm). P r o d u c t Identification. The tritiated 3,4-dihydroxyphenylalanine product of the reaction is separated from tritiated tyrosine by partition with alumina by a modification of the method of Nagatsu et al., ~8 and then identified by paper and thin-layer chromatography. The reaction is prepared as before, except that solution 5 containing an inhibitor of L-aromatic amino acid decarboxylase (EC 4.1.1.26) is used rather than solution 2, and reactions are prepared in heavy-walled glass conical centrifuge tubes. After incubation, 20 f~l of solution 9 is added and the reaction is terminated o0S. Udenfriend, in "Fluorescence Assay in Biology and Medicine," p. 154. Academic Press, New York, 1962. ~IF. H. Schneider and C. N. Gillis, Biochem. Pllarmacol. 14, 623 (1965).

788

ISOLATION AND CULTURE OF CELLS

[77]

by the addition of 2 ml of solution 10 at 3 o. After 15 minutes at 3 o, tubes are centrifuged at 10,000 g for 15 minutes. The supernatant fluid is added with stirring to a beaker containing 10.5 ml of alumina gel prepared as described above; the pH is adjusted to 8.5 with 3 N NH4OH, the mixture is stirred for 5 minutes at 24 ° and then allowed to stand at room temperature without stirring for an additional 10 minutes. After the alumina has settled, the supernatant fluid is decanted and the gel is transferred to disposable Pasteur pipette plugged with glass wool and washed with 30 ml of H20. Catechol reaction products are eluted with 2 ml of solution 6. The radioactivity of a small portion of the eluate is determined (approximately 78% of dihydroxyphenylalanine added to alumina is recovered in the column eluate); the remainder of the eluate is evaporated to dryness under a stream of nitrogen gas and then dissolved in 50 ~1 of H20. Portions of the product then are subjected to ascending paper and thin-layer chromatography using the following systems: 1. Paper chromatography with Whatman No. 1 paper. Solvent I (Armstrong et al.62): 1-butanol:acetic acid:H20 (12: 3:5). The Ri values of 3,5-dihydroxyphenylalanine and tyrosine are 0.21 and 0.46, respectively. 2. Thin-layer chromatography using Eastman 6064 thin-layer plates. Solvent II (Nybom 63) : Methylethylketone:formic acid :H20 (24: 1:6). The R I values of 3,5-dihydroxyphenylalanine and tyrosine are 0.13 and 0.30, respectively. Solvent III (Aures et al.64): Ethyl acetate:acetic acid:H20 (15: 15:10). The R~ values of 3,5-dihydroxyphenylalanine and tyrosine are 0.73 and 0.86, respectively. Solvent IV (Johnson and Boukma6~): 1-butanol: 1 N acetic acid: absolute ethanol (35: 10:10). The RI values of 3,5-dihydroxyphenylalanine and tyrosine are 0.20 and 0.35, respectively. Catechols are located by spraying developed and dried chromatograms with solution 7. Chromatograms then are placed in a stream of warm air until dry and then viewed under UV light. Chromatograms are sprayed with ninhydrin solution (Sigma Chem. Co., St. Louis, Missouri) to detect tyrosine. When dry, chromatograms are cut into 1 × 1 cm (paper) or 0.5 × l cm (thin-layer) strips, each is placed in a scintillation vial with 1 ml of solution 8 and 10 ml of solution 4, and radioactivity is determined with a liquid scintillation counter. ~2M. D. Armstrong, K. N. F. Shaw, and P. E. Wall, J. Biol. Chem. 218, 293 (1956). e3N. Nybom, Physiol. Plantarum 17, 434 (1964). D. Aures, R. Fleming, and R. Hakanson, J. Chrornatogr. 33, 480 (1968). ~G. A. Johnson and S. J. Boukma, Anal. Biochem. 18, 143 (1967).