Preliminary
homogenate of bovine mammary gland. Since EAC cells originate from a mammary carcinoma of the mouse, this finding is suggestive of the tissue specificity characteristic for a chalone. Further experiments to elucidate the nature of the inhibitory compounds in the UF are in progress.
notes
399
were transferred by somatopetal intra-axonal transport and accumulated in corresponding dorsal root ganglia neurons. The tracers were retained during the preparation of cell suspensions. The accumulation of the fluorescent tracer Evans blue was quantitated by cytofluorometric measurements on individual neurons.
Nerve cells can incorporate macromolecules at the axon terminals, e.g. horseradish peroxidase (HRP), which can then be transported in the axon to the perikaryon References in the somatopetal direction [ 11.For evalua1. Lehmann, W, Graetz, H, Schiitt, M & Langen, P, tion of the biological meaning of this pheActa biol med Germ 36 (1977) K 43. 2. Holley, R W, Nature 258 (1975) 487. nomenon it would be of importance to study 3. Gospodarowicz, D & Moran, J S, Ann rev bioif physiological or pathological alterations them 45 (1976) 531. 4. Teng, M H, Bartholomew, J C & Bissell, M J, Proc in the axon terminal result in quantitative natl acad sci US 73 (1976) 3173. changes in the nerve cell body accumula5. Nissley, S P, Rechler, M H, Moses, A C, Short, P A & Podskalny, J M, Endocrinology 101 (1977) tion of macromolecules. For such studies 708. quantitative cytochemical techniques, 6. Ktllema, J A & Linebough, B E, Biochim biophys acta 475 (1977) 74. which permit analysis of individual cells, 7. Shields, R, Nature 267 (1977) 308. 8. de Asua, L J, O’Farell, M, Bennett, D, Clingan, D are desirable, also since they enable the & Rudland, P, Nature 265 (1977) 151. detection of possible heterogeneities within 9. Negelein, E, Leistner, I & Jahnchen, L, Acta biol the cell populations with respect to uptake med Germ 16 (1966) 372. 10. Documenta Geigy, Wiss Tabellen, 1st edn, p. 724. and transport. For quantitative cytochemiJ R Geigy AG, Pharma, Base1(1969). cal studies it is much more favourable to 11. Sokawa, Y, Watanabe, Y & Kawade, Y, Nature 268 (1977) 236. use single whole cells than cells in tissue sections. Received June 22, 1978 Revised version received October 27, 1978 Although HRP has been extremely useful Accepted November 6, 1978 as a tracer for combined light and ultrastructural studies, it is for several reasons less suitable for quantitation. The enzyme Printed in Sweden Copyright @ 1979 by Academic Press, Inc. histochemical reaction is difficult to standAll rights of reproduction in any form reserved OOl4-4827/79/04399-05$02.00/O ardize and results in the formation of very dark masses of granules with a heteroCytofluorometric quantitation of retrograde geneous distribution in the cell. The abaxonal transport in single dorsal root ganglia sorbance of such material is difftcult to neurons quantitate with sufficient accuracy. A LENNART ENERBACK, KRISTER KRISTENSfluorescent probe is much more suitable for SON and LENNART A. PERSSON, Deparrment of quantitation in studies of this type. The Pathology II, University of Linkiiping, S-581 85 Lingreat advantage with cytofluorometry lies kiiping, Sweden in its independence of the distribution of Summary. A technique is described by which neurons from mouse dorsal root ganglia can be dispersed in fluorescent material in the cell and its single-cell suspensions suitable for quantitative cytomoderate demands on instrumentation. chemical analyses. The neurons were intact as conUnder ideal conditions the total fluorestrolled by trypan blue exclusion test, and the cell size distribution of the dispersed neurons corresponded to cence emitted from a cell is directly proporthat of untreated, intact ganglia. Horseradish pertional to its total content of fluorescent maoxidase and Evans blue applied to cut sciatic nerve, Exp Cell Rcs 119 (1979)
400
Preliminary notes
terial. The present study describes a method by which neuronal incorporation of a tracer substance can be measured by cytofluorometry on single cells from spinal root ganglia. Materials and Methods of cell suspensions. About 60-day-old female Swiss albino mice were used (Anticimex AB, Stockholm). The mice were sacrificed by decapitation after ether anaesthesia and the lumbar dorsal root ganglia L IV and L V were dissected, cut in 4-6 pieces, incubated for 60 min in 200 ~12 % collagenase (Type I, Sigma) w/v in a buffer containing Hepes (Sigma) 100 mmol/l, NaCl 66 mmol/l, KCI 6.7 mmol/l, and 1% bovine serum albumin (fraction V, Sigma). The pH value was adjusted to 7.5 with 1 M NaOH. The specimens were shaken every 15 min. After incubation 80 ~1 90 % calf serum in phosphate buffer was added and the suspensions were left in an ice-bath for 5 min. The cell suspensions were then centrifuged for 10 min at 800 rpm at 4°C and rinsed twice in the medium. Throughout the preparatory procedure siliconized glassware were used. To test the permeability of the nerve cell plasma membrane, isolated neurons were suspended in an albumin-free medium and trypan blue was added to a final concentration of 0.45 % [2]. Cell counting and sizing. The number of neurons obtained from each ganglion was counted in a FuchsRosenthal hemocytometer. The mean diameter of single neurons was measured with a Vickers imagesplitting eyepiece (Vickers Ltd, York) on air-dried suspensions, fixed in 2.5% glutaraldehyde in phosphate buffer, stained with toluidine blue and mounted in Diatexe. The mean diameter was calculated from two perpendicular measurements on each cell. For comparison, similar cell diameters were measured on enlarged micrographs of 1 pm thick epon embedded sections from intact ganglia. Only neurons with a nucleolus in the plane of section were measured.
Preparation
Tracer technique for somatopetal
axonal transport.
In order to test the ability of the dispersed neurons to retain an exogenous tracer substance 0.25 mg HRP (Type II, Sigma) was applied as dry powder to cut sciatic nerveat the mid:ihigh level of-5 mice during ether anaesthesia, 24 h prior to dispersion. HRP activity was demonstrated in sediments of isolated cells fixed in 2.5% glutaraldehyde in phosphate buffer and incubated in 3’,3-diaminobenzihine - and H,O, [3]. Epon embedded cells pelleted in agar gel was used for ultrastructural localization of HRP. As fluorogenic tracer we used 10~1 of 10% Evans blue (Merck, Darmstadt) w/v in saline [4, 51, similarly applied to cut sciatic nerve. Twenty-four hours later non-fixed dispersed single neurons were mounted in HEPES buffer on glass slides and 175consecutive cells were analysed for fluorescence. The contralateral dorsal root ganglion of the same treated mouse was dissected and single neurons from these ganglia served as control. Cyrofluorometry. An automated cytofluorometer was used, to be described in detail elsewhere. The instrument is based on a Leitz MPVII microscopic E.rp Cd Rrs 119 (19791
photometer equipped with electromagnetic shutters and controlled by a minicomputer (Digital Equipment PDP 8e). The instrument incorporated a dual beam arrangement for correction of lamp variations due to arc instability [6]. A glass slide was used as a beam splitter and an image of the field diaphragm formed on the surface of a silicon detector. The correction was performed with an electronic divider, making a quotient between the fluorescence signal and the signal from the detector. Green light was used for activation of the red Evan’s blue fluorescence in neurons. Activating light from a Xenon burner was filtered through a Schott BG 36 glass filter and a combination of a short pass interference filter and a barrier filter with maximum transmission between 530 and 560 nm. A dichroic mirror with 50% transmission and absorption at 580 nm was used and the fluorescent light filtered with a combination of a short pass and a barrier filter transmitting between 580 and 610 nm. We used a fluorescent uranyl standard and all measured values were normalized to “uranyl units” as described previously [7]. The neurons to be measured were identified with phase contrast microscopy. For fluorescence measurements, field and measuring diaphragms had diameters of 70 and 50 pm in the object plane.
Results and Discussion Several methods to isolate pure fractions of neurons from CNS for biochemical studies have been used previously [S-12]. In the present study employing measurements on single, microscopically identified cells, pure neuronal fractions were not required although we were aiming at a high degree of biochemical and structural integrity of the dispersed cells. A specific demand on the method was that the permeability of the plasma membrane for larger molecules should remain intact after cell dispersal. After a series of preliminary experiments the described technique, using a medium previously used for isolation of intact liver cells [2] was found to give satisfactory results. On the average, 2000 neurons could be recovered from each ganglion and 95.1% (S.E.M.=0.07; 21 observations) of the cells excluded trypan blue. Although the value of the trypan blue exclusion test as an indicator of viability can be seriously questioned, it appears to be a reliable indicator of the permeability state of the plasma membrane for substances of similar or
Preliminary notes
401
Figs l-3. Single neurons containing fluorescent Evans blue granules cfig. I) and granules of HRP reaction product Ifig. 2). One+m thick section of Epone.t;b.tiyd neurons stained with toluidine blue @g. 3).
Fig. 4. Electron micrograph of neuron-containing HRP
larger molecular weight, i.e. 960 [13]. The procedure did not involve a selective loss of any particular cell size, since the size distribution of the dispersed single neurons was bimodal and comparable to that of intact ganglia (fig. 5) [14].
It has previously been observed that 24 h after HRP application to cut sciatic nerve HRP accumulates in the majority of neurons in sections from corresponding dorsal root ganglia [ 151.HRP could also be demonstrated in the majority of both small and
reaction product in vesicles in the cytoplasm. x 20 000.
Exp Cell Res II9 (1979)
402
Preliminary notes
40
I
r
A
30 i
Fig. 5. Abscissa: cell diameters (pm); ordinnte: no. of cells. Histogram showing bimodal distribution of cell sizes of (A) isolated neurons; (B) in sections of dorsal root
large dispersed single neurons in the present study (fig. 2). Ultrastructurally, HRP reaction product occurred in membrane-bound vesicles in the cytoplasm of the neurons (fig. 4). The neurons were well preserved as observed by light microscopy (fig. 3) and ultrastructurally the plasma membrane appeared intact with evaginations and invaginations of the surface [ 141. These experiments therefore shows that the protein tracer HRP was retained in the neurons during the dispersion procedure. Evans blue also accumulated in the neurons and appeared as red fluorescent granules in the cytoplasm (fig. 1). In neurons from the contralateral ganglia, a faint diffuse fluorescence was seen which appeared to be of approximately the same intensity as the autofluorescence common to most cells and tissues. For this experiment, any interference by autofluorescent lipofuscin granules was reduced by the use of young mice, whose nerve cells contain few such granules. The majority of the ipsilateral ganglia neurons exhibited higher Exp Cell Res 119 (1979)
Fig. 6. Abscissa: fluorescence intensity (many1 units); ordinate: no. of cells. Histograms showing distribution of fluorescent intensities in isolated neurons. Evans blue was applied to the cut sciatic nerve and fluorescence recorded in ipsilateral ganglion cells (II). The contralateral ganglion cells served as controls (A).
fluorescence intensity than neurons of the contralateral (control) ganglia (fig. 6). The fluorescence of the latter probably represented autofluorescence and possibly minor amounts of blood-borne tracers. The difference in fluorescence intensity between the ipsi- and contralateral ganglia neurons thus reflected the somatopetal transport of the tracer. The mean fluorescence of the control neurons was 22.4 (S.D. 13.7) and mean fluorescence of ipsilateral ganglia neurons 105.5 (S.D. 106). As seen in fig. 6B the distribution of the fluorescence in ipsilateral ganglia neurons was positively skewed. A preliminary test using probably charts showed that the values were well
Preliminary
adjusted to a normal distribution after logarithmic transformation. The population did not exhibit any signs of bimodality with respect to fluorescent dye content. This study was supported by grants from the Swedish Medical Research Council No. 04480 and 02235.
References 1. Kristensson, K, Ann rev pharmacol toxic01 18 (1978) 97. 2. Segler, P, Methods in cell biology (ed D M Prescott) 1st edn, vol. 13, p. 30. Academic Press, New York (1976). Graham, R C & Karnovsky, M J, J histochem cytochem 14 (1%6) 291. Kristensson, K, Acta neuropath 16 (1970) 293. Kuypers, H Cl J M, Catsman-Berrevoets, C E & Padt, R E, Neurosci lett (1977) 127. Rundquist, I, Enerblck, L & Gustafsson, B, VI Ennineerina foundation conference on automated cyt;logy (1578). Abstract. I. Enerback, L, Berlin, G, Svensson, I & Rundquist, I, J histochem cytochem 24 (1976) 1231. 8. Hvden, H & Pigeon, A. J neurochem 6 (1960) 57. 9. Blomstrand, C& Hamberger, A, J neurochem 16 (1%9) 1401. 10. Norton, W T & Poduslo, S E, Science 167 (1970) 1144. 11. Capps-Covey, P & McIlwain, D L, J neurochem 2s (1975) 517. 12. Poduslo, S E & McKhann, G M, Brain res 132 (1977) 107. 13. Bhvyan, B K, Loughman, B E, Fraser, T J & Day, K J, Exp cell res 97 (1976) 275. 14. Lieberman, A R, The peripheral nerve (ed D N Landon) p. 188. Chapman Hall, London (1976). 15. Kristensson, K & Olssoh, Y, J neurocyt 4 (1975) 653. Received July 7, 1978 Revised version received October 23, 1978 Accepted November 7, 1978
Printed in Sweden Copyright @ 1919 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/04403-04u)2.00/0
Protein phosphokinase activity of rat liver nuclear membrane RANDOLPH C. STEER, MICHAEL J. WILSON and KHALIL AHMED.’ Toxicolo~v Research Laboratory, Department of’Laborato~wMedicine and Pathology, University of Minnesota, Veterans Adminstration Medical Center, Minneapolis, MN 55417, USA
The presence of protein phosphokinase activity in a purified nuclear-membrane preparation from adult rat liver was demonstrated by measuring the in-
Summary.
notes
403
corporation of 32P from -Q*P-ATP into endogenous nuclear-membrane oroteins as well as into the exogenous protein substrates, dephosphophosvitin (DPV) and lvsine-rich histone (LRH). The activitv of this enzyme toward DPV was 60 times greater than that toward LRH. CAMP and cGMP did not appear to affect the phosphorylation of endogenous-membrane proteins. ,
I
r
Much attention has been focused on protein phosphorylation as a possible regulatory control mechanism in cellular function, In this context the presence of protein phosphokinases at numerous subcellular locations has been reported [l-4]. Accordingly, specialized roles for phosphoproteins localized at various sites within the cell have been suggested [4-91. Although numerous studies have resulted in implicating cell membrane phosphoprotein phosphorylation reactions in a variety of cellular activities [4-91, to date no such study of the presence of protein phosphokinase reactions in the nuclear membrane has been documented. This might be due to the difficulty in obtaining that cellular component in an uncontaminated form, i.e., without chromatin or other subcellular components present. Thus, it seemed warranted to obtain a sufficiently pure preparation of nuclear membrane and to determine as a first step, what, if any, protein phosphokinase reactions are associated with it. In the present brief report we describe the presence of protein phosphokinase reactions in a purified nuclear membrane preparation. Its physiological function remains to be established. Materials
and Methods
In each experiment, adult (350 g) male rats (ARS Sprague-Dawley, Madison Wise.) were used. Lysinerich histone from calf thymus and deoxyribonuclease I from bovine pancreas were obtained from Worthing1 To whom reprint requests should be addressed; Toxicology Research Laboratory, Veterans Administration Medical Center, 4801 E. 54th Street, Minneapolis, MN 55417, USA. Exp Cell Res 119(1979)
homogenate of bovine mammary gland. Since EAC cells originate from a mammary carcinoma of the mouse, this finding is suggestive of the tissue specificity characteristic for a chalone. Further experiments to elucidate the nature of the inhibitory compounds in the UF are in progress.
notes
399
were transferred by somatopetal intra-axonal transport and accumulated in corresponding dorsal root ganglia neurons. The tracers were retained during the preparation of cell suspensions. The accumulation of the fluorescent tracer Evans blue was quantitated by cytofluorometric measurements on individual neurons.
Nerve cells can incorporate macromolecules at the axon terminals, e.g. horseradish peroxidase (HRP), which can then be transported in the axon to the perikaryon References in the somatopetal direction [ 11.For evalua1. Lehmann, W, Graetz, H, Schiitt, M & Langen, P, tion of the biological meaning of this pheActa biol med Germ 36 (1977) K 43. 2. Holley, R W, Nature 258 (1975) 487. nomenon it would be of importance to study 3. Gospodarowicz, D & Moran, J S, Ann rev bioif physiological or pathological alterations them 45 (1976) 531. 4. Teng, M H, Bartholomew, J C & Bissell, M J, Proc in the axon terminal result in quantitative natl acad sci US 73 (1976) 3173. changes in the nerve cell body accumula5. Nissley, S P, Rechler, M H, Moses, A C, Short, P A & Podskalny, J M, Endocrinology 101 (1977) tion of macromolecules. For such studies 708. quantitative cytochemical techniques, 6. Ktllema, J A & Linebough, B E, Biochim biophys acta 475 (1977) 74. which permit analysis of individual cells, 7. Shields, R, Nature 267 (1977) 308. 8. de Asua, L J, O’Farell, M, Bennett, D, Clingan, D are desirable, also since they enable the & Rudland, P, Nature 265 (1977) 151. detection of possible heterogeneities within 9. Negelein, E, Leistner, I & Jahnchen, L, Acta biol the cell populations with respect to uptake med Germ 16 (1966) 372. 10. Documenta Geigy, Wiss Tabellen, 1st edn, p. 724. and transport. For quantitative cytochemiJ R Geigy AG, Pharma, Base1(1969). cal studies it is much more favourable to 11. Sokawa, Y, Watanabe, Y & Kawade, Y, Nature 268 (1977) 236. use single whole cells than cells in tissue sections. Received June 22, 1978 Revised version received October 27, 1978 Although HRP has been extremely useful Accepted November 6, 1978 as a tracer for combined light and ultrastructural studies, it is for several reasons less suitable for quantitation. The enzyme Printed in Sweden Copyright @ 1979 by Academic Press, Inc. histochemical reaction is difficult to standAll rights of reproduction in any form reserved OOl4-4827/79/04399-05$02.00/O ardize and results in the formation of very dark masses of granules with a heteroCytofluorometric quantitation of retrograde geneous distribution in the cell. The abaxonal transport in single dorsal root ganglia sorbance of such material is difftcult to neurons quantitate with sufficient accuracy. A LENNART ENERBACK, KRISTER KRISTENSfluorescent probe is much more suitable for SON and LENNART A. PERSSON, Deparrment of quantitation in studies of this type. The Pathology II, University of Linkiiping, S-581 85 Lingreat advantage with cytofluorometry lies kiiping, Sweden in its independence of the distribution of Summary. A technique is described by which neurons from mouse dorsal root ganglia can be dispersed in fluorescent material in the cell and its single-cell suspensions suitable for quantitative cytomoderate demands on instrumentation. chemical analyses. The neurons were intact as conUnder ideal conditions the total fluorestrolled by trypan blue exclusion test, and the cell size distribution of the dispersed neurons corresponded to cence emitted from a cell is directly proporthat of untreated, intact ganglia. Horseradish pertional to its total content of fluorescent maoxidase and Evans blue applied to cut sciatic nerve, Exp Cell Rcs 119 (1979)
400
Preliminary notes
terial. The present study describes a method by which neuronal incorporation of a tracer substance can be measured by cytofluorometry on single cells from spinal root ganglia. Materials and Methods of cell suspensions. About 60-day-old female Swiss albino mice were used (Anticimex AB, Stockholm). The mice were sacrificed by decapitation after ether anaesthesia and the lumbar dorsal root ganglia L IV and L V were dissected, cut in 4-6 pieces, incubated for 60 min in 200 ~12 % collagenase (Type I, Sigma) w/v in a buffer containing Hepes (Sigma) 100 mmol/l, NaCl 66 mmol/l, KCI 6.7 mmol/l, and 1% bovine serum albumin (fraction V, Sigma). The pH value was adjusted to 7.5 with 1 M NaOH. The specimens were shaken every 15 min. After incubation 80 ~1 90 % calf serum in phosphate buffer was added and the suspensions were left in an ice-bath for 5 min. The cell suspensions were then centrifuged for 10 min at 800 rpm at 4°C and rinsed twice in the medium. Throughout the preparatory procedure siliconized glassware were used. To test the permeability of the nerve cell plasma membrane, isolated neurons were suspended in an albumin-free medium and trypan blue was added to a final concentration of 0.45 % [2]. Cell counting and sizing. The number of neurons obtained from each ganglion was counted in a FuchsRosenthal hemocytometer. The mean diameter of single neurons was measured with a Vickers imagesplitting eyepiece (Vickers Ltd, York) on air-dried suspensions, fixed in 2.5% glutaraldehyde in phosphate buffer, stained with toluidine blue and mounted in Diatexe. The mean diameter was calculated from two perpendicular measurements on each cell. For comparison, similar cell diameters were measured on enlarged micrographs of 1 pm thick epon embedded sections from intact ganglia. Only neurons with a nucleolus in the plane of section were measured.
Preparation
Tracer technique for somatopetal
axonal transport.
In order to test the ability of the dispersed neurons to retain an exogenous tracer substance 0.25 mg HRP (Type II, Sigma) was applied as dry powder to cut sciatic nerveat the mid:ihigh level of-5 mice during ether anaesthesia, 24 h prior to dispersion. HRP activity was demonstrated in sediments of isolated cells fixed in 2.5% glutaraldehyde in phosphate buffer and incubated in 3’,3-diaminobenzihine - and H,O, [3]. Epon embedded cells pelleted in agar gel was used for ultrastructural localization of HRP. As fluorogenic tracer we used 10~1 of 10% Evans blue (Merck, Darmstadt) w/v in saline [4, 51, similarly applied to cut sciatic nerve. Twenty-four hours later non-fixed dispersed single neurons were mounted in HEPES buffer on glass slides and 175consecutive cells were analysed for fluorescence. The contralateral dorsal root ganglion of the same treated mouse was dissected and single neurons from these ganglia served as control. Cyrofluorometry. An automated cytofluorometer was used, to be described in detail elsewhere. The instrument is based on a Leitz MPVII microscopic E.rp Cd Rrs 119 (19791
photometer equipped with electromagnetic shutters and controlled by a minicomputer (Digital Equipment PDP 8e). The instrument incorporated a dual beam arrangement for correction of lamp variations due to arc instability [6]. A glass slide was used as a beam splitter and an image of the field diaphragm formed on the surface of a silicon detector. The correction was performed with an electronic divider, making a quotient between the fluorescence signal and the signal from the detector. Green light was used for activation of the red Evan’s blue fluorescence in neurons. Activating light from a Xenon burner was filtered through a Schott BG 36 glass filter and a combination of a short pass interference filter and a barrier filter with maximum transmission between 530 and 560 nm. A dichroic mirror with 50% transmission and absorption at 580 nm was used and the fluorescent light filtered with a combination of a short pass and a barrier filter transmitting between 580 and 610 nm. We used a fluorescent uranyl standard and all measured values were normalized to “uranyl units” as described previously [7]. The neurons to be measured were identified with phase contrast microscopy. For fluorescence measurements, field and measuring diaphragms had diameters of 70 and 50 pm in the object plane.
Results and Discussion Several methods to isolate pure fractions of neurons from CNS for biochemical studies have been used previously [S-12]. In the present study employing measurements on single, microscopically identified cells, pure neuronal fractions were not required although we were aiming at a high degree of biochemical and structural integrity of the dispersed cells. A specific demand on the method was that the permeability of the plasma membrane for larger molecules should remain intact after cell dispersal. After a series of preliminary experiments the described technique, using a medium previously used for isolation of intact liver cells [2] was found to give satisfactory results. On the average, 2000 neurons could be recovered from each ganglion and 95.1% (S.E.M.=0.07; 21 observations) of the cells excluded trypan blue. Although the value of the trypan blue exclusion test as an indicator of viability can be seriously questioned, it appears to be a reliable indicator of the permeability state of the plasma membrane for substances of similar or
Preliminary notes
401
Figs l-3. Single neurons containing fluorescent Evans blue granules cfig. I) and granules of HRP reaction product Ifig. 2). One+m thick section of Epone.t;b.tiyd neurons stained with toluidine blue @g. 3).
Fig. 4. Electron micrograph of neuron-containing HRP
larger molecular weight, i.e. 960 [13]. The procedure did not involve a selective loss of any particular cell size, since the size distribution of the dispersed single neurons was bimodal and comparable to that of intact ganglia (fig. 5) [14].
It has previously been observed that 24 h after HRP application to cut sciatic nerve HRP accumulates in the majority of neurons in sections from corresponding dorsal root ganglia [ 151.HRP could also be demonstrated in the majority of both small and
reaction product in vesicles in the cytoplasm. x 20 000.
Exp Cell Res II9 (1979)
402
Preliminary notes
40
I
r
A
30 i
Fig. 5. Abscissa: cell diameters (pm); ordinnte: no. of cells. Histogram showing bimodal distribution of cell sizes of (A) isolated neurons; (B) in sections of dorsal root
large dispersed single neurons in the present study (fig. 2). Ultrastructurally, HRP reaction product occurred in membrane-bound vesicles in the cytoplasm of the neurons (fig. 4). The neurons were well preserved as observed by light microscopy (fig. 3) and ultrastructurally the plasma membrane appeared intact with evaginations and invaginations of the surface [ 141. These experiments therefore shows that the protein tracer HRP was retained in the neurons during the dispersion procedure. Evans blue also accumulated in the neurons and appeared as red fluorescent granules in the cytoplasm (fig. 1). In neurons from the contralateral ganglia, a faint diffuse fluorescence was seen which appeared to be of approximately the same intensity as the autofluorescence common to most cells and tissues. For this experiment, any interference by autofluorescent lipofuscin granules was reduced by the use of young mice, whose nerve cells contain few such granules. The majority of the ipsilateral ganglia neurons exhibited higher Exp Cell Res 119 (1979)
Fig. 6. Abscissa: fluorescence intensity (many1 units); ordinate: no. of cells. Histograms showing distribution of fluorescent intensities in isolated neurons. Evans blue was applied to the cut sciatic nerve and fluorescence recorded in ipsilateral ganglion cells (II). The contralateral ganglion cells served as controls (A).
fluorescence intensity than neurons of the contralateral (control) ganglia (fig. 6). The fluorescence of the latter probably represented autofluorescence and possibly minor amounts of blood-borne tracers. The difference in fluorescence intensity between the ipsi- and contralateral ganglia neurons thus reflected the somatopetal transport of the tracer. The mean fluorescence of the control neurons was 22.4 (S.D. 13.7) and mean fluorescence of ipsilateral ganglia neurons 105.5 (S.D. 106). As seen in fig. 6B the distribution of the fluorescence in ipsilateral ganglia neurons was positively skewed. A preliminary test using probably charts showed that the values were well
Preliminary
adjusted to a normal distribution after logarithmic transformation. The population did not exhibit any signs of bimodality with respect to fluorescent dye content. This study was supported by grants from the Swedish Medical Research Council No. 04480 and 02235.
References 1. Kristensson, K, Ann rev pharmacol toxic01 18 (1978) 97. 2. Segler, P, Methods in cell biology (ed D M Prescott) 1st edn, vol. 13, p. 30. Academic Press, New York (1976). Graham, R C & Karnovsky, M J, J histochem cytochem 14 (1%6) 291. Kristensson, K, Acta neuropath 16 (1970) 293. Kuypers, H Cl J M, Catsman-Berrevoets, C E & Padt, R E, Neurosci lett (1977) 127. Rundquist, I, Enerblck, L & Gustafsson, B, VI Ennineerina foundation conference on automated cyt;logy (1578). Abstract. I. Enerback, L, Berlin, G, Svensson, I & Rundquist, I, J histochem cytochem 24 (1976) 1231. 8. Hvden, H & Pigeon, A. J neurochem 6 (1960) 57. 9. Blomstrand, C& Hamberger, A, J neurochem 16 (1%9) 1401. 10. Norton, W T & Poduslo, S E, Science 167 (1970) 1144. 11. Capps-Covey, P & McIlwain, D L, J neurochem 2s (1975) 517. 12. Poduslo, S E & McKhann, G M, Brain res 132 (1977) 107. 13. Bhvyan, B K, Loughman, B E, Fraser, T J & Day, K J, Exp cell res 97 (1976) 275. 14. Lieberman, A R, The peripheral nerve (ed D N Landon) p. 188. Chapman Hall, London (1976). 15. Kristensson, K & Olssoh, Y, J neurocyt 4 (1975) 653. Received July 7, 1978 Revised version received October 23, 1978 Accepted November 7, 1978
Printed in Sweden Copyright @ 1919 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/04403-04u)2.00/0
Protein phosphokinase activity of rat liver nuclear membrane RANDOLPH C. STEER, MICHAEL J. WILSON and KHALIL AHMED.’ Toxicolo~v Research Laboratory, Department of’Laborato~wMedicine and Pathology, University of Minnesota, Veterans Adminstration Medical Center, Minneapolis, MN 55417, USA
The presence of protein phosphokinase activity in a purified nuclear-membrane preparation from adult rat liver was demonstrated by measuring the in-
Summary.
notes
403
corporation of 32P from -Q*P-ATP into endogenous nuclear-membrane oroteins as well as into the exogenous protein substrates, dephosphophosvitin (DPV) and lvsine-rich histone (LRH). The activitv of this enzyme toward DPV was 60 times greater than that toward LRH. CAMP and cGMP did not appear to affect the phosphorylation of endogenous-membrane proteins. ,
I
r
Much attention has been focused on protein phosphorylation as a possible regulatory control mechanism in cellular function, In this context the presence of protein phosphokinases at numerous subcellular locations has been reported [l-4]. Accordingly, specialized roles for phosphoproteins localized at various sites within the cell have been suggested [4-91. Although numerous studies have resulted in implicating cell membrane phosphoprotein phosphorylation reactions in a variety of cellular activities [4-91, to date no such study of the presence of protein phosphokinase reactions in the nuclear membrane has been documented. This might be due to the difficulty in obtaining that cellular component in an uncontaminated form, i.e., without chromatin or other subcellular components present. Thus, it seemed warranted to obtain a sufficiently pure preparation of nuclear membrane and to determine as a first step, what, if any, protein phosphokinase reactions are associated with it. In the present brief report we describe the presence of protein phosphokinase reactions in a purified nuclear membrane preparation. Its physiological function remains to be established. Materials
and Methods
In each experiment, adult (350 g) male rats (ARS Sprague-Dawley, Madison Wise.) were used. Lysinerich histone from calf thymus and deoxyribonuclease I from bovine pancreas were obtained from Worthing1 To whom reprint requests should be addressed; Toxicology Research Laboratory, Veterans Administration Medical Center, 4801 E. 54th Street, Minneapolis, MN 55417, USA. Exp Cell Res 119(1979)
