We have used the 'Fast Protein Liquid Chromatography' system manufactured by Pharmacia to study the screening and separation of restriction endonucleases.
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BIOCHEMICAL SOCIETY TRANSACTIONS
Fast protein liquid chromatography We have used the ‘Fast Protein Liquid Chromatography’ system manufactured by Pharmacia to study the screening and separation of restriction endonucleases. The number of supports available commercially are currently limited to those for gel filtration, ion-exchange, chromatofusing and reversed-phase applications. For the initial isolation of restriction endonucleases from crude extracts we have found that 1 ml of quaternary aminoethyl ‘Mono Q’ strong anion-exchange column is ideal. All the restriction enzymes we studied were eluted between 0.2 and 0.6M-KCl. This has enabled us to get to up a rapid screening programme for novel type-I1 restriction endonuclease activities. The profile of enzyme activities from an extract of a cyanobacterium Nostoc SA, incubated with lambda DNA, shows four separate restriction enzymes of different specificity. These enzyme fractions were separately digested with other DNA substrates. These and other experiments lead us to conclude that the enzymes were NspSAI (C-YC-G-R-G), NspSAII (G-G-T-N-A-C-C), NspSAIII (C-C-AT-G-G), NspSAIV (G-G-A-T-C-C) and were isoschizomers of AvaI, BstEII, NcoI and BarnHI, respectively. The distribution of cytosines and guanines in each cutting site is interestingly consistent. We have found that the purity of these and other enzymes after a single Mono Q column step is sufficient not only for characterization of their specificity on a series of substrates but also to carry out analysis of the termini of the cutting sites using a modified form of M 13 sequencing. The usefulness of f.p.1.c. in optimizing separations is immediately clear when scaling up these columns. Not only are preparative columns available but also the separation conditions for the macro-bead column equivalent may be easily found using the f.p.1.c. counterpart. Further research is planned to extend the range of
restriction specificities using this screening procedure. The support of the Medical Research Council, the Wolfson Foundation, P and S Biochemicals and the University of Liverpool is gratefully acknowledged. Bickle, T. A., Purotta, V. & Imber, R. (1977)Nucleic Acids Res. 4,
2561-25 72 Butler,
P. E., Fairhurst, D.
& Beynon, R. J . (1984)Anal. Biochem.
141,494-498 Craven, D. B., Harvey, M. J., Lowe, C. R. & Dean, P. D. G. (1974) Eur. J. Biochem. 41,329-333 Dean, P. D. G., Johnson, W. S. & MIddle, F. A. (1985) Affinity Chromatography,A Practical Approach, IRL Press, Oxford Farooqui, A. A. (1980)J. Chromatogr. 184,335-345 Larsson, P. O., Glad, M., Hansson, L., Mansson, M-O., Ohlson, S. & Mosbach, K. (1983)Adv.Chromatogr. 21,41-85 Lowe, C. R. & Dean, P. D. G. (1974) Affinity Chromatography, Wiley, New York Lowe, C. R., Glad, M., Larsson, P a . , Ohlson, S., Small, D. A. P., Atkinson, A. & Mosback, K. (1981)J. Chromatogr. 215, 303-
316 Ohlson, S., Hansson, L., Larsson, P.-0. & Mosbach, K. (1978)FEBS. Lett. 93,5-9 Porath, J. (1979)Colloq. Inst. Nut. Sante Rech. Med. 86,17-36 Sassenfeld, H. & Brewer, S. (1984)Biotechnology 2,76-81 Small, D. A. P., Atkinson, A. & Lowe, C. R. (1981)J. Chromatogr.
216,175-190 Small, D. A. P., Atkinson, A. & Lowe (1983)in Affinity Chromatography and Biological Recognition (Chaiken, I. M.,Wilcheck, M & Parikh, I., eds.), pp. 267-268, Academic Press, New York Smith, H. 0. & Wilcox, K. W. (1970)J. Mol. Biol. 51,379-391 Subramanian, S. (1984)CRCOit. Rev. Biochem. 16,169-205 Vijayalakshmi, M. A. (1983) in Affinity Chromatography and Biological Recognition (Chaiken, I. M., Wilcheck, M. & Parikh, I., eds.), pp. 269-273, Academic Press, New York Walters, R. R. (1983) in Affinity Chromatography and Biological Recognition (Chaiken, I. M., Wilcheck, M. & Parikh, I., eds.), pp. 261-264, Academic Press, New York
Part 2: Recent Advances in High-Performance Liquid Chromatographic Analysis of Small Molecules
Application of high-performance liquid chromatography with electrochemical detection to the study of neurotransmitters in vivo In recent years extensive detail has been generated on anatomical, biochemical and pharmacological aspects of the central neurotransmitter control of blood pressure under normal and stressful conditions (Saavedra ef al., 1978; Ross et al., 1983). However, there is only circumstantial evidence linking release of specific monoamine neuroAbbreviations used: ECD, electrochemical detection; 5-HT, 5transmitters with this control. In order to correlate monohydroxytryptamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 5HIAA, 5-hydroxyindoleacetic acid; Ru 24969,5-methoxy-3(1,2,3,6- amine release with a particular function the transmitter tetrahydro4-pyridinyl-lH-indole; 8-OHDPAT, 8-hydroxy-2-(di-n- must be measured in vivo. Recent improvements in, and propylamine) tetralin. development of, methods that can monitor endogenous *To whom correspondence should be addressed. neurotransmitter release and metabolism from related brain 1985
C. ROUTLEDGE and C. A. MARSDEN* Department of Physiology and Pharmacology, Nottingham Medical School, Nottingham NG7 2UH, U.K.
1059
613th MEETING, CARDIFF regions now enable a correlation to be made between extracellular monoamine levels and function. One approach to the measurement of neurotransmitter release and metabolism involves intracerebral perfusion, in which brain perfusate samples are collected and assayed for their transmitter and metabolite content using specific and highly sensitive analytical techniques. An example of this approach is intracerebral dialysis combined with h.p.1.c. and electrochemical detection (ECD) (Zetterstrom er a[., 1983; Ungerstedt, 1984). This technique was used in the present study as it has the advantage of being discriminative and highly sensitive. The large size of the probe limits study to the larger brain areas such as the frontal cortex. However, modification and refinement of the dialysis technique has allowed the measurement of neurotransmitter release from the hypothalamus. The hypothalamus presents certain problems for this technique in that the distribution of monoamines is very localized and thus selective regional measurements need to be made. It is possible to measure relative monoamine levels in different hypothalamic regions and to monitor changes in them after stimulation of brain stem areas innervating the hypothalamus. We have used intracerebral dialysis to investigate autoregulation of 5-hydroxytryptamine (5-HT) release in the frontal cortex and to study the adrenergic involvement of the hypothalamic regulation of blood pressure. The dialysis loops were made from flexible cellulose tubing (diam. 250pm, M, cut-off 5000). Lengths (2 cm) of 25 G stainless steel cannulae were glued onto both ends of a 4.2 cm length of dialysis tubing, leaving an exposed length of tubing of 2 mm at the centre of the cannulae. A piece of fine fishing line (80pm diam.) was threaded into the dialysis tubing and positioned inside the lumen of the exposed tubing. A piece of polythene tubing was attached to one of the steel cannula; the other was bent to an angle of 140" and connected to a perfusion system. The tubing was perfused until thoroughly wet and then bent into a loop; the fishing line prevented the loop from kinking. The loop was immersed into physiological saline before implantation. Implantation into the frontal cortex and hypothalamus was carried out according to standard stereotaxic techniques under chloral hydrate (600 mg/kg intraperitoneally) or halothane anaesthesia. The loop was perfused at a rate of 0.7pl/min, and after a 1 h pre-collection period samples of perfusate were collected every 20 or 30min. These were assayed for noradrenaline, adrenaline, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) by h.p.1.c.-ECD. Monoamines and their metabolites were separated by either reverse-phase chromatography (Spherisorb 50DS 2 column, mobile phase 0.1 M-
acetate/citrate, pH 4.6, containing 10% methanol) for DOPAC, 5-HT and 5-HIAA or ion-pair reverse-phase (mobile phase 0.1 M-NaH2P04 pH 3.6, 0.1 mM-EDTA, 0.1 mM-sodium octonyl sulphonic acid and 9% methanol) for noradrenaline, adrenaline, dopamine, DOPAC and 5-HIAA. In order to correlate the amount of compounds in the perfusate with extracellular levels, the recovery of these compounds across the dialysis membrane must be known. To estimate recovery dialysis loops were perfused in virro at a rate of 0.7 and lpl/min and placed in physiological saline containing the monoamines at 10-6M. The amount of substance in the perfusate was compared with the amount outside the tubing and expressed as percentage recovery. Each loop was thus calibrated, and the extracellular levels of the amines and their metabolites estimated. However, it is not clear to what extent recovery determined in virro reflects the recovery of the amines from the brain environment, and as yet there is no method for determining recovery in vivo. The applicability of the dialysis probe to measurement of amines in vivo was initially tested in the frontal cortex, striatum and ventricles of the rat. Table 1 shows basal levels in the striatum, frontal cortex and cerebrospinal fluid of the anaesthetized rat and in the frontal cortex of the freely moving rat. Basal extracellular levels were found to be higher in the conscious animal; this emphasizes the usefulness of the dialysis technique for monitoring amine levels in the freely moving animals, and indicates that the anaesthetic is decreasing amine release and metabolism. Monoamine metabolite levels were present in very much higher concentrations than the parent amines. Intracerebral dialysis was then used to monitor levels of these amines and metabolites after administration of two putative 5-HTI receptor agonists. Binding and electrophysiological studies have suggested the existence of multiple 5-HT receptors (Peroutka & Snyder, 1979), which have been further subdivided to ~ - H T I A~, - H T I Band 5-HTz receptors, and there is now interest in linking these subreceptor types to specific 5-HT-induced physiological and pharmacological responses. Two putative 5-HT1 receptor agonists have recently been developed, 5-methoxy-3( 1,2,3,6tetrahydro-4-pyridinyl)-l€j-indole(RU 24969) (Hunt & Oberlander, 1981) and 8-hydroxy-2-(di-n-propylamine) tetralin (8-OHDPAT) (Arvidsson et al., 1981), which is a specific agonist at 5-HTIA receptors. Studies with RU 24969 and 8-OHDPAT have suggested that the 5-HT1 receptor is the S-HT autoreceptor. The effects of administration of RU 24969 and 8-OHDPAT were monitored in the frontal cortex of anaesthetized rats. RU 2496 (10mg/ kg intraperitoneally) decreased extracellular 5-HT (Fig. la)
Table 1. Estimated extracellular concentrations o f 5-HT, 5-HIAA and DOPAC in the striatum, frontal cortex and ventricles o f anaesthetized and freely moving rats
The values have been corrected for recovery in vitro through the dialysis tubing (see text for details). Extracellular concn. (M) Area of brain
...
Compound
DOPAC 5-HT 5-HIAA
Vol. 1 3
Frontal cortex
Striatum
3.4 x 10-6 4.5 x 1.0 x 10-6
Anaesthetized
Freely moving
5.0 X lo-' 6.2 X lo-* 4.3 x 10-7
6.7 x lo-* 1.2 x 6.1 x
Ventricles
4.5 x 10-7 -
3.9 x 10-7
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BIOCHEMICAL SOCIETY TRANSACTIONS
80 -
70 8-OHDPAT (n = 6 )
' 6050 -
40 30 -
,I
20 -
2o 6o 1o01w60
Time (min)
3b0
2'0
60 100 140 t80'220~260300340
Time (min)
Fig. 1. Effect of (a) RU 24969 ( I 0 mgfkg intraperitoneally) and ( b ) 8-OHDPAT (0.32 mglkg subcutaneously) on extracellular 5-HT levels in the rat frontal cortex measured b y in tracere bra1 dialysis The results are expressed as a percentage (mean % f S . E . M . ) of the sample immediately pre-injection ( 100%).
dialysis. The dialysis loops were implanted into the hypothalamus and the C1 area was electrically stimulated for 30 min by using modified SNE 100 (Clarke Electromedical Equipment) concentric needle electrodes (2 V, 1 ms rectangular pulses at 40 Hz, for 10 s every 30 s). Hypothalamic samples were collected every 30min and assayed for adrenaline, noradrenaline, dopamine, DOPAC and 5-HIAA by h.p.1.c.-ECD. Blood pressure was recorded from a cannulated demoral artery. The C1 region was stimulated for 30min after a 120min stabilization period, and the effects were monitored for a further 120 min. Extracellular levels of adrenaline, noradrenaline, dopamine, DOPAC and 5-HIAA in the posterior h pothalamus were estimated to be 6.7 x lO-'M, 7.9 x 10-2M, 1.6 x lO-'M and 2.5 x 1 0 - 6 M respectively. Again an important feature was the low levels of amines compared with the amine metabolites. Stimulation of the C1 region of the ventrolateral
(a)
-5
120
-
100
-
C, region
Control area
v
-'z2 80
E
2
and 5-HIAA levels to 46% and 33% respectively of preinjection control values 200 min after administration. This effect was also observed in the freely moving rat, though the effect was more pronounced, again indicating that the anaesthesia was altering 5-HT release and metabolism. 8OHDPAT (0.32 mg/kg subcutaneously) decreased extracellular 5-HT (Fig. l b ) and 5-HIAA levels by 70% and 60% respectively in the frontal cortex of anaesthetized rats 100 min post-injection. The fall in extracellular 5-HIAA and 5-HT after administration of RU 24969 supports the view from studies in uitro (Middlemiss, 1984) that 5-HTI receptors are involved in the autoregulation of 5-HT release and metabolism. The decrease in 5-HT release and metabolism after 8-OHDPAT also suggests autoreceptor function though it needs to be determined whether this reduction in 5-HT metabolism is a primary effect via a 5-HT autoreceptor or secondary to an effect on a post-synaptic 5-HT receptor on another neuron. The hypothalamus has been implicated in the role of blood pressure regulation and is thought to play a part in the onset and maintenance of hypertension (Thornton er d.,1984). Evidence has shown that the central noradrenergic and adrenergic neuron pathways are strikingly similar to central pathways involved in cardiovascular regulation (Hokfelt et al., 1974), in particular those pathways originating in the ventrolateral medulla ascending to the hypothalamus and descending to the spinal cord. Adrenaline and noradrenaline are postulated to be the transmitters present with the adrenaline cell bodies located in the vasopressor C1 region of the ventrolateral medulla projecting to the hypothalamus and the intermediolateral column of the spinal cord. The noradrenaline cell bodies are located in the caudal ventrolateral medulla (Al region), which is vasodepressor, and they project to the hypothalamus. Studies have been carried out to try and correlate a change in monoamine levels in the hypothalamus with changes in blood pressure after stimulation of the C1 region of the ventrolateral medulla. Hypothalmic extracellular monoamine levels were measured by intracerebral
P
--
~1
60
T T
-
m
(b1
120
-
h
5?
2
v
loo-
2
2
80-
2
-
a .E!
60
-
40
-
20
-
m + -t
2
1
O L Pre- During
Pre- During
Fig. 2. Pre- and post-stimulation extracellular adrenaline levels (a) and mean arterial pressure ( b )
Either the C1 region or an area outside but close to the C 1 (control) was stimulated for 30 rnin (see text for details). The adrenaline value in the 30 min pre-stimulation dialysis sample is compared with the sample collected during the stimulation period. With the mean arterial pressure the mean of the values obtained during these two periods is given. Results are given as means f S.E.M.(n = 5). Note the significant increase in adrenaline and mean arterial pressure during stimulation of the C1 region and the absence of change during stimulation of the control region. 1985
1061
613th MEETING, CARDIFF medulla resulted in an increase in extracellular adrenaline levels in the posterior hypothalamus and a simultaneous increase in mean arterial pressure. Extracellular adrenaline increased to 56% of pre-stimulation control values when a corresponding blood pressure rise of 47.7 mmHg (+ 65%) was observed. Neither adrenaline or mean arterial pressure increased when the stimulating electrodes were outside the C1 region (Fig. 2). No change was seen in hypothalamic extracellular levels of noradrenaline, dopomine, DOPAC and 5-HIAA during the stimulation period; however, there was a significant increase in extracellular noradrenaline levels in the post-stimulation perfusate sample. This delayed increase in noradrenaline may be the result of a reflex response to the increase in blood pressure and not due to the electrical stimulus spreading to the A, region. The increase in adrenaline levels in the hypothalamus after stimulation of the C1 region gives support to the evidence for an adrenergic pressor pathway from the rostra1 ventrolateral medulla to the hypothalamus. The lack of correlation between an increase in extracellular levels of noradrenaline, dopamine, DOPAC and 5-HIAA and mean arterial pressure after C1 stimulation suggests that the increase in mean arterial pressure during electrical stimulation of the C1 region relates to a specific increase in adrenaline levels. However, the C, region also innervates the spinal cord (Ross et al., 1981). An adrenergic pathway descends from the C1 region to the intermediolateral column of the spinal cord. Stimulation of the C, region may also increase adrenaline levels in the intermediolateral
column causing a corresponding increase in mean arterial pressure. Further studies are necessary to determine whether the pressor responses are mediated through an adrenergic pathway to the hypothalamus or by a direct pathway to the spinal cord. We thank The Wellcome Trust for financial support. C.A.M. is Wellcome Senior Lecturer and C.R. is an S.E.R.C. C.A.S.E. student in collaboration with ICI plc. Arvidsson, L. E., Hacksall, U., Nilsson, J . L. G., Hjorth, S., Carlsson, A., Lindberg, F., Sanchez, D. & Wikstrom, H . (1981) J. Med. Chem. 24,921-927 Hokfelt, T., Fuxe, K., Goldstein, M. & Johansson, 0. (1974) Brain Res. 66,235-251 Hunt, P. F. & Oberlander, C. (1981) in Serotonin - Current Aspects of Neurochemistry and Function (Haber, B., ed.), pp. 547-562, Plenum Press, New York Middlemiss, D. N. (1984) 14th C.I.N.P. Congr. 657 Peroutka, S.J. & Snyder, S. H . (1979)Mol. Pharmacol. 16, 687 Ross, C. A., Armstrong, D. M., Ruggiero, D. A,, Pickel, V. M., Joh, T. H. & Reis, D. J. (1981) Neurosci. Lett. 25, 257 Ross, C. A., Ruggiero, D. A , , Joh, T. H., Par, D. H. & Reis, D. J. (1983) Brain Res. 273,356-361 Saavedra, J. M., Grobecker, H. & Axelrod, J. (1978) Circulation Res. 42,529-534 Thorton, S . N., De Beaurepaine, R. & Nicolaidis, S. (1984) Brain Res. 2 9 9 , l - 7 Ungerstedt, U. (1984) in Measurement of Neutotransmitter Release in uivo (Marsden, C. A. ed.), pp. 81--106, John Wiley, Chichester Zetterstrom, T., Sharp, T., Marsden, C. A . & Ungerstdet, U. (1983) J. Neurochem. 41,1769-1773
The application of high-performance liquid chromatography to the purification of oligosaccharides containing neutral and acetamido sugars ELIZABETH F. HOUNSELL, NICOLA J. JONES and MARK S. STOLL Applied Immunochemistry Research Group, M.R.C. Clinical Research Centre, Watford Road, Harrow HA1 3UJ, Middx. U.K. Oligosaccharides which occur in body fluids, such as milk and urine, or as the carbohydrate chains of secreted and cell-surface glycoproteins are usually found as complex mixtures of closely related molecules. Their purification involves the separation, not only of molecules of different size and composition, but also of isomers which vary in linkage position or anomeric configuration. Our studies on the structural characterization and assignment of the antigenicities of cell-surface carbohydrates has been made possible by the finding that the oligosaccharides of interest are also present on secreted glycoproteins such as the mucins of human meconium (Wood et al., 1979; Gooi et al., 1981, 1983a, b , Hounsell et al., 1985) and in milk (Gooi et al., 1981; Hounsell et al., 1 9 8 1 ~ Gooiet ; al., 1985), which can be obtained in relatively large amounts. However, because of the heterogeneity of the carbohydrate structures, efficient chromatographic methods are required for purification before structural and antigenic elucidation. H.p.1.c. has proved to be of great value in oligosaccharide purification and analysis because of the wide range of adsorbents and solvent systems available, the relatively short separation time and the high yield, which allow Abbreviations used: ODs, silica bonded with octadecyl groups; APS, silica bonded with aminopropyl groups; h.p.t.l.c., highperformance thin-layer chromatography.
Vol. 13
several different chromatographic separations to be carried out. More than one chromatographic system has been shown to be necessary for purification of oligosaccharide isomers (Hounsell et al., 1981a,b, 1984,1985) and thus we have investigated the h.p.1.c. separation of native and acetylated oligosaccharide alditols by using silica, silica bonded with octadecyl (ODS) or with aminopropyl (APS) groups and an anion-exchange resin.
The combined use of h.p.1.c. on reverse-phase and aminebonded silica column supports in oligosaccharide purification Reverse-phase chromatography of oligomers of glucose (Cheetham et al., 1981) and of N-acetylglucosamine (Blumberg et al., 1982) with ODS column packings gave a separation with an increased retention time for oligomers with higher M, values. However, when oligosaccharides containing both neutral and acetamido sugars were chromatographed on ODS (Cheetham & Dube, 1983; Dua & Bush, 1983, Hounsell et al., 1984a), a separation by size was not achieved as oligosaccharides having a higher acetamido/ neutral sugar ratio have a longer retention time due to the relative hydrophobicity of acetamido groups compared with hydroxyl. Size separation of oligosaccharides containing neutral and acetamido sugars can be achieved by h.p.1.c. on aminobonded silica using either acetonitrile/water (Ng Ying Kin & Wolfe. 1980; Boersma et al., 1981; Mellis & Baenziger 1981, Turco, 1981; Warren et al., 1983) or acetonitrile/ phosphate buffers (Bergh et al., 1981, 1983; Parante e t a l . , 1984) and silica eluted with solvents containing soluble amine modifiers (Aitzetmuller, 1978; Wheals & White,
