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A simplified method to prepare soluble brain proteins for electrophoresis
SHORT
119
COMMUNICATIONS
enzymes seen in myopathic processes. Before any deductions can be made, an accurate idea of the normal activity of these enzymes is needed. The figures here presented indicate two features. (a) There is a difference in overall enzyme activity between paraspinal and limb musculature in those enzymes studied. (b) Characteristic distribution patterns for these enzymes occur. Both these features tend to support the view that the demands made by the two types of muscle on their enzyme systems may be different and comparative studies of isozyme representation may perhaps further reflect this. It seems pertinent to remark that if variations occur in activity of enzymes in different muscle groups, then it is important to state the source of the muscle, together with the reported activity when recording enzyme assays. We gratefully acknowledge the untiring enthusiasm of Mr. Werner Jansen and his assistants in the technical processing of this material. The work was supported by the Muscular Dystrophy Association of Canada. Neuromuscular University Edmonton, I 2 3 4 =j 6
Research
G. MONCKTON
Unit,
Hospital, University Alberta (Canada)
T. NIHEI
of Alberta,
J, A. SIBLEY AND A. L. LEHNINGER, J. Natl. Cancer Inst., 9 (1949) 141. G. SCHAPIRA, J. C. DREYFUS AND F. SCHAPIRA, Semaine Hop. Paris, 29 (1953) 1917. K. TAD.\, Y. WATANABE AND H. CHIKACKA, Tohoku J. Exptl. Med., 7513 (1961) 299. I. T. OLIVER, Biochem. J., 61 (1955) 116. F. WROBEWSKI AND J. S. LADUE, Sot. Exptl.. Biol. Med., go (1955) 210. B. LUDVIGSEN, J. Lab. Clin. Med., 61 (1963) 329.
Received June qth,
1965 Clin. Chim. Acta,
13 (1966)
117-119,
A simplified method to prepare soluble brain proteins for electrophoresis Nervous tissue soluble proteins are usually prepared directly with saline’ or buffered saline extraction a-7 or with the same solvents, after defatting the tissue with Bloor mixtures-11. To obtain only cytoplasmic proteins, isotonic sucrose (0.25 M) has also been used1z-14. In this paper, a simplified method is described for the preparation of soluble nervous tissue proteins for electrophoretic analysis, consisting in centrifugation at high speed. The supematant obtained and recentrifuged is ready for running. Clin. Chim. Acta,
13 (1966)
11g-121
120
SHORT
METHODS
AND
COMMUNICATIONS
MATERIALS
3-4 mm thick slices of (human) brain were cut with a lancet and rapidly washed in saline. 1-1.5 g of nervous tissue were homogenized for 3 min at 2’ with a small amount of quartz powder in a Potter-Elvehjem homogenizer, consisting of a polypropylene ultracentrifuge the homogenate examined homogenate
tube and a “Delrin” (DuPont) in a contrast-phase microscope
was centrifuged
at 104,000 x g for 60 min at
pestle. A small amount of showed no intact cells. The 2’
in a high-speed
centrifuge
Fig. 1. Grey matter of the temporal lobe. The upper part of the microslide shows the ‘3 brain protein fractions. Below, the electrophoretic pattern of a normal human serum as a control. (Amido Schwarz B 19 staining; 25 min running at 120 V).
(M.S.E.
40) and five layers
were obtained.
The second
layer,
composed
of protein
solution, was isolated and recentrifuged at ro,oooxg for IO min at 2”; the clear supernatant was used for the electrophoretic run. 0.01 ml of protein extract were electrophoretically analyzed, according to the Wieme’s agar gel technique15, in parallel to a normal human serum; the samples were run in triplicate; of each sample, three homogenates were prepared. The plates, stained according to Uriel and Scheideggerlg, were then read by an “Extinktionsschreiber Zeiss II”” apparatus modified for microslides by the firm as suggested by us. RESULTS
AND
DISCUSSION
The nitrogen content (biuret)iT of the protein extracts varied according to the brain regions; for grey matter it was 1.80-2.20 gob in the parietal and temporal lobes, 2.20-2.30 g% in the frontal lobe, 2.50-2.90 g% in the occipital lobe ; for white matter 2.60-3.30 g% in the frontal lobe, 2.40-2.45 g% in the temporal lobe, 3.30-3,70 g% in the parietal lobe, 2.20-2.90 g% in the occipital lobe; for cerebellum 2.95-3.70 g%. Clin.
Chim.
Acta,
13 (1966)
II~-IZI
SHORT
121
COMMUNICATIONS
Using this procedure, to the zones investigated.
the number of the fractions Reproducibility
varied from 13 to 16, according
of the extraction
runnings were always more than 95% (PLo.05). This dilution of the tissue proteins, so that extracts of higher In addition, neither physical nor chemical factors of save those connected with the ultracentrifugation at may be thought
that, by this technique,
method
and that of the
extraction technique avoids protein content are obtained. denaturation are introduced, low temperature. Finally, it
also less concentrated
protein
fractions
can
be detected. Institute of Neurobiology and Pathological Anatomy “A. Verga” of the Provincial Psychiatric Institutes of Milano, Via Ippocrate 45, Milano, and Institute of General Pathology, University of Milan0 (Italy)
A. ALLEGRANZA P. CANEVINI P. MOCARELLI
I G. I
Received
July
r6th,
1965 Clin. Chim. Acta, 13 (1966) II~--121
lsolement et composition chimique de deux macroglobulines de Waldenstrijm La fraction des euglobulines isolee du serum de malades atteints de macroglobulinemie de Waldenstrijm renferme un composant majeur antigeniquement rattachk au groupe des yM-globulines (Ig M) du serum humain. Sa purification a CtC facilitee par l’introduction de la chromatographie de gel-filtration sur gel de Sephadex G200. Nous rapportons l’isolement et l’etude de deux macroglobulines pathologiques. PR$PARATIONDES
MACROGLOBULINES
Le serum dilue au dixieme avec de l’eau distill&e est dialyse pendant 48 h a 4” contre dix volumes de tampon phosphate 0.005 M de pH 6.0. Le precipite forme en Clin. Chim. Acta,
13 (1966) I~I--125
119
COMMUNICATIONS
enzymes seen in myopathic processes. Before any deductions can be made, an accurate idea of the normal activity of these enzymes is needed. The figures here presented indicate two features. (a) There is a difference in overall enzyme activity between paraspinal and limb musculature in those enzymes studied. (b) Characteristic distribution patterns for these enzymes occur. Both these features tend to support the view that the demands made by the two types of muscle on their enzyme systems may be different and comparative studies of isozyme representation may perhaps further reflect this. It seems pertinent to remark that if variations occur in activity of enzymes in different muscle groups, then it is important to state the source of the muscle, together with the reported activity when recording enzyme assays. We gratefully acknowledge the untiring enthusiasm of Mr. Werner Jansen and his assistants in the technical processing of this material. The work was supported by the Muscular Dystrophy Association of Canada. Neuromuscular University Edmonton, I 2 3 4 =j 6
Research
G. MONCKTON
Unit,
Hospital, University Alberta (Canada)
T. NIHEI
of Alberta,
J, A. SIBLEY AND A. L. LEHNINGER, J. Natl. Cancer Inst., 9 (1949) 141. G. SCHAPIRA, J. C. DREYFUS AND F. SCHAPIRA, Semaine Hop. Paris, 29 (1953) 1917. K. TAD.\, Y. WATANABE AND H. CHIKACKA, Tohoku J. Exptl. Med., 7513 (1961) 299. I. T. OLIVER, Biochem. J., 61 (1955) 116. F. WROBEWSKI AND J. S. LADUE, Sot. Exptl.. Biol. Med., go (1955) 210. B. LUDVIGSEN, J. Lab. Clin. Med., 61 (1963) 329.
Received June qth,
1965 Clin. Chim. Acta,
13 (1966)
117-119,
A simplified method to prepare soluble brain proteins for electrophoresis Nervous tissue soluble proteins are usually prepared directly with saline’ or buffered saline extraction a-7 or with the same solvents, after defatting the tissue with Bloor mixtures-11. To obtain only cytoplasmic proteins, isotonic sucrose (0.25 M) has also been used1z-14. In this paper, a simplified method is described for the preparation of soluble nervous tissue proteins for electrophoretic analysis, consisting in centrifugation at high speed. The supematant obtained and recentrifuged is ready for running. Clin. Chim. Acta,
13 (1966)
11g-121
120
SHORT
METHODS
AND
COMMUNICATIONS
MATERIALS
3-4 mm thick slices of (human) brain were cut with a lancet and rapidly washed in saline. 1-1.5 g of nervous tissue were homogenized for 3 min at 2’ with a small amount of quartz powder in a Potter-Elvehjem homogenizer, consisting of a polypropylene ultracentrifuge the homogenate examined homogenate
tube and a “Delrin” (DuPont) in a contrast-phase microscope
was centrifuged
at 104,000 x g for 60 min at
pestle. A small amount of showed no intact cells. The 2’
in a high-speed
centrifuge
Fig. 1. Grey matter of the temporal lobe. The upper part of the microslide shows the ‘3 brain protein fractions. Below, the electrophoretic pattern of a normal human serum as a control. (Amido Schwarz B 19 staining; 25 min running at 120 V).
(M.S.E.
40) and five layers
were obtained.
The second
layer,
composed
of protein
solution, was isolated and recentrifuged at ro,oooxg for IO min at 2”; the clear supernatant was used for the electrophoretic run. 0.01 ml of protein extract were electrophoretically analyzed, according to the Wieme’s agar gel technique15, in parallel to a normal human serum; the samples were run in triplicate; of each sample, three homogenates were prepared. The plates, stained according to Uriel and Scheideggerlg, were then read by an “Extinktionsschreiber Zeiss II”” apparatus modified for microslides by the firm as suggested by us. RESULTS
AND
DISCUSSION
The nitrogen content (biuret)iT of the protein extracts varied according to the brain regions; for grey matter it was 1.80-2.20 gob in the parietal and temporal lobes, 2.20-2.30 g% in the frontal lobe, 2.50-2.90 g% in the occipital lobe ; for white matter 2.60-3.30 g% in the frontal lobe, 2.40-2.45 g% in the temporal lobe, 3.30-3,70 g% in the parietal lobe, 2.20-2.90 g% in the occipital lobe; for cerebellum 2.95-3.70 g%. Clin.
Chim.
Acta,
13 (1966)
II~-IZI
SHORT
121
COMMUNICATIONS
Using this procedure, to the zones investigated.
the number of the fractions Reproducibility
varied from 13 to 16, according
of the extraction
runnings were always more than 95% (PLo.05). This dilution of the tissue proteins, so that extracts of higher In addition, neither physical nor chemical factors of save those connected with the ultracentrifugation at may be thought
that, by this technique,
method
and that of the
extraction technique avoids protein content are obtained. denaturation are introduced, low temperature. Finally, it
also less concentrated
protein
fractions
can
be detected. Institute of Neurobiology and Pathological Anatomy “A. Verga” of the Provincial Psychiatric Institutes of Milano, Via Ippocrate 45, Milano, and Institute of General Pathology, University of Milan0 (Italy)
A. ALLEGRANZA P. CANEVINI P. MOCARELLI
I G. I
Received
July
r6th,
1965 Clin. Chim. Acta, 13 (1966) II~--121
lsolement et composition chimique de deux macroglobulines de Waldenstrijm La fraction des euglobulines isolee du serum de malades atteints de macroglobulinemie de Waldenstrijm renferme un composant majeur antigeniquement rattachk au groupe des yM-globulines (Ig M) du serum humain. Sa purification a CtC facilitee par l’introduction de la chromatographie de gel-filtration sur gel de Sephadex G200. Nous rapportons l’isolement et l’etude de deux macroglobulines pathologiques. PR$PARATIONDES
MACROGLOBULINES
Le serum dilue au dixieme avec de l’eau distill&e est dialyse pendant 48 h a 4” contre dix volumes de tampon phosphate 0.005 M de pH 6.0. Le precipite forme en Clin. Chim. Acta,
13 (1966) I~I--125