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R. Harris, E. Ukaejiofo (1970)
Tissue Typing Using a Routine One‐Step Lymphocyte Separation ProcedureBritish Journal of Haematology, 18
B. Davis (1964)
DISC ELECTROPHORESIS – II METHOD AND APPLICATION TO HUMAN SERUM PROTEINS *Annals of the New York Academy of Sciences, 121
O. Lowry, N. Rosebrough, A. Farr, R. Randall (1951)
Protein measurement with the Folin phenol reagent.The Journal of biological chemistry, 193 1
(1970)
DIFCO ( I 969) Tissue culture and virus propagation. D$eo Technical Information
(1969)
Purification of mitogenic proteins derived from Phaseolus vulgaris : isolation of potent and weak PHAs possessing mitogenic activity
V. Räsänen, T. Weber, R. Gräsbeck (1973)
Crystalline kidney-bean leucoagglutinin.European journal of biochemistry, 38 1
(1970)
The cytotoxic activity of isoantibodies in mice
pol.vacvalumide gel. Note 75. LKB Productor, Uppsala, Sweden. phenol reagent
Hospital Management Committee , Oswestry . U . K . pol . vacvalumide gel . Note 75 . LKB Productor , Uppsala , Sweden . phenol reagent
973) Crystalline kidney bean leucoagglutinin
(1970)
Determination of tryptophan in the protein
M. Gaitonde, T. Dovey (1970)
A rapid and direct method for the quantitative determination of tryptophan in the intact protein.The Biochemical journal, 117 5
(1975)
Kinetics of DNA synthesis and cell proliferation of in oitro stimulated human leucocytes . 7 th Meeiing of ihe European Stud ? ! Group for Cell Proli $ eration
95 1) Protein measurement with the Folin-PHARMACIA (1974) Biomedical Research Product Guide. p. 1 I. Pharmacia Fine Chemicals AB
(1965)
Disc electrophoresis . 11 . Method and application to human serum proteins
L. Allen, R. Svenson, S. Yachnin (1969)
Purification of mitogenic proteins derived from Phaseolus vulgaris: isolation of potent and weak phytohemagglutinins possessing mitogenic activity.Proceedings of the National Academy of Sciences of the United States of America, 63 2
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Orthopaedic LKB APPLICATION NOTE (1973) LKB 21 17 Multiphor-I. Ana!vtical thin-layer gel elecirofocussing in LOWRY
J. S M I T HA N D J . C . A L L E N Biochemistrjj Research Unit, North East Wales Institute of Higher Education, Clwyd, Wales (Received 20 November 1977: revision accepted 23 February 1978) ABSTRACT Three commercially available purified phytohaemagglutinin (PHA) preparations have been examined both chemically and biologically. Marked differences were observed in both composition and mitogenic effect on lymphocytes; the existence of at least two distinct mitogenic fractions in PHA was confirmed. A number of commercial preparations of PHA have become available for use as mitogens in lymphocyte cultures. Since there is no cross-standardization of biological activity between these preparations, workers have used their own discretion about which to use and whether further purification is desirable. Three commercial purified PHA preparations have been examined, and marked biological and chemical differences have been found. MATERIALS AND METHODS PHA preparations were purchased as freeze-dried powders: Difco âPâ (purified grade) from Difco Ltd, Central Avenue, West Molesey, Surrey, U.K.; Wellcome âPurifiedâ from Wellcome Reagents Ltd, Beckenham, Kent, U.K.; and âLeucoagglutininâ from Pharmacia Fine Chemicals Ltd, Uppsala, Sweden. The manufacturerâs data on these âpurifiedâ grades of PHA indicate that they are specifically prepared for their mitogenic action and that erythrocyte agglutinin activity is either absent or very low (Difco, 1969; Pharmacia, 1974). Tissue culture Rabbit peripheral blood lymphocytes were isolated from whole peripheral blood using a Ficoll-Triosil density gradient (Harris & Ukaejiofo, 1970), and were resuspended in RPMI 1940 tissue culture medium (Flow), supplemented with 10% (v/v) foetal calf serum, penicillin-streptomycin (100 i d 1 0 0 mg per ml), L-glutamine (8.0 mM) and sodium bicarbonate (0.2% w/v). Cells were counted and used immediately. Assays were performed on 5 x lo4 Correspondence: Dr J . C. Allen, Kelsterton College, Connahâs Quay, Clwyd CH5 4 B R . Wales. 0008-8730/78/110&0659$02.00 ~ 1 9 7 Blackwell Scientific Publications 8 659 C. J. Smith and J. C. Allen or lo5 cells suspended in 0.2 ml of medium in Falcon Microtest I1 culture plates. Weighed samples of PHA were dissolved in culture medium with dilutions such that a maximum of 10 p1 of PHA solution was added to each of the cultures. Cultures were incubated at 37°C in an atmosphere of 7% CO,, 10% 0, and 83% N, for 72 hr; tritiated thymidine ([3HlTdR, 0.5 pCi/well. Radiochemical Centre, Amersham, U.K.) was added to each culture 24 hr prior to termination by precipitation of the acid-insoluble fraction with 5% (v/v) ice-cold trichloroacetic acid. Radioactivity was monitored using a Nuclear Chicago mark I1 liquid scintillation counter. Cytotoxicity was estimated by the dye exclusion test (Gover & OâGorman, 1956). The above procedure was adopted after a preliminary comparison of 1, 2 and 3 day culture times had shown an optimal stimulation on day 3. Protein determinations were by the method of Lowry et al. (1 95 1) and gel electrophoresis was performed using the method of Davis (1965). Gels were stained in 1% (w/v) amido black in 7% (v/v) acetic acid. Cellulose acetate electrophoresis was carried out over 2 hr in 0.05 M barbitone buffer (pH 8.6), using a current density of 1.0 mA/cm strip width. Strips were stained in amido black (0-25 g in 3% (w/v) trichloroacetic acid in methanol, 100 ml) for 1 hr and destained in methanol :glacial acetic acid (9 : 1 v/v). Amino acid analyses were carried out using a Locarte single column automatic amino acid analyser. Samples were hydrolysed in 6.0 M HCI (Aristar Grade) containing 5% (v/v) 0.1 M thiodiglycol in uucuo for 24 hr at 100OC. Cysteine was determined separately after hydrolysis in performic acid for 2 hr at 0°C. Tryptophan was assayed by the method of Gaitonde & Dovey (1970). Isoelectric points were determined by isoelectric focusing, using the LKB âMultiphorâ flat-bed apparatus over pH range 3.5-9.5 (LKB Application Note, 1973). RESULTS AND DISCUSSION The incorporation of [3H]TdR into DNA is used widely as a measure of DNA synthesis (Feinendegen, 1967). Bernheim & Mendelsohn have shown that this is a valid method for the assessment of lymphocyte mitosis when the cell concentration is between 1 and 5 x lo5 per ml (Bernheim & Mendelsohn, 1975). Experiments to determine the PHA concentration needed for optimum mitogenic response showed marked differences between the preparations (Table 1). Above this optimum, the PHA became increasingly cytotoxic as assessed by the popular dye exclusion test. Whereas Pharmacia âLeucoagglutininâ was the most active in terms of weight required to produce maximal stimulation, both Difco âPâ and Wellcome âPurifiedâ induced greater absolute stimulation. The existence of at least two mitogenic components was thus suggested, and was substantiated by jnvestigation of the chemical properties of the preparations. Their protein contents exhibited large variations (Table 2). even among the three âpurifiedâ materials; the value for a âreagentâ grade material, Difco âMâ, is included for comparison. Although electrophoresis on cellulose acetate (Fig. 1) showed that âLeucoagglutininâ and Wellcome âPurifiedâ each consisted of only one band, their mobilities were different. Difco âPâ contained both these components, plus a further âslowerâ one whose contribution to its total biological activity is unknown. Isoelectric focusing confirmed that the PI of âLeucoagglutininâ is 5.1 (Rasanen, Weber, & Grasbeck, 1973). Although Wellcome âPurifiedâ separated into six bands (PI = 4.1,4.4,4.9, 5.6, 7.3 and 7.7), there was no component with PI = 5.1, thus substantiating the electrophoretic results. Difco âPâ gave numerous bands. Commercial PHA preparations TABLE I . Dose-response of lymphocytes to commercial P H A preparations I âHITdR incorporation (dimin) PHA concentration (pgiml) âLeucoagglutininâ Wellcome âPurifiedâ Difco âPâ 1.25 2.50 3.75 5 .O 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 2653 8736 17619 16480 789 I 4140 9625 32589 26158 12976 1543 1 15944 8500 7480 699 I 2247 Mean dose-response (s.d. not greater than lSU/o) using triplicate 3 day cultures of rabbit peripheral blood from three animals, with LâHITdR added for the last 24 hr. Number of cells used = 5 x 10â cells per well. For culture conditions see text. Control value, in absence of PHA = 432.5 TABLE2. Protein content of commercial PHA preparations Percentage protein Sample Pharmacia âLeucoagglutininâ Wellcome âPurifiedâ Difco âPâ Difco âMâ Estimation by the Lowry method, using bovine serum albumin as a standard. The amino acid analyses (Table 3) are comparable with those of Hurn (1966), who noted unexplained differences between his own preparations. The content of the neutral and acidic residues were similar in all, but the Wellcome âPurifiedâ contained a higher proportion of basic residues. These results also accord with the view that Difco âPâ consists largely of a mixture of the two components of the other PHAs. since the values from Difco âPâ of all but three amino acids are intermediate between those of âLeucoagglutininâ and Wellcome âPâ. Allen, Svenson & Yachnin (1969) separated Difco âPâ into seventeen bands by polyacrylamide gel electrophoresis, and showed that chromatography on C M Sephadex gave several components with varied degrees of mitogenic activity. The results cited here confirm C.J. Smith and J. C. Allen FIG.1. Cellulose acetate electrophoresis of commercial purified PHA preparations, in 0.05 M sodium barbiturate. pH 8.6. A current density of 1.0 mA/cm strip width was maintained for 2 hr. Strips were stained in amido black. (0) origin; (D) Difco âPâ;(W) Wellcome âPurifiedâ; (L) Pharmacia âLeucoagglutininâ. TABLE Amino acid composition of commercial purified PHA preparations 3. Composition (g amino acid per 100 g) Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Isoleucine Tyrosine Phenylalanine Tryptophan Pharmacia âLeucoagglutininâ Difco âPâ Wellcome âPurifiedâ No methionine or cysteine residues were detected. the existence of at least two mitogenic components in PHA, demonstrate that different commercial preparations contain different mitogens. and serve as a caveat to those who assume the homogeneity of âpurifiedâ PHA. Commercial PHA preparations ACKNOWLEDGMENT The authors thank Nicholas International Ltd for financial support.
Cell Proliferation – Wiley
Published: Nov 1, 1978
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