1424
BIOCHEMICAL SOCIETY TRANSACTIONS
I
I
I
10
20
50
1 I+ 100 NSB
Serum basic protein (ng/ml) Fig. 2. Assay of serum myelin basic protein
.,
Details of the assay are given in the text. Standards contained human myelin basic protein in either pooled normal human serum or blood-bank plasma. Inhibition of tracer binding by human myelin basic protein; NSB, non-specific tracer binding in the absence of specific antisera. 0,V , Inhibition of tracer binding by serial dilutions of sera from 64ng/ml; V , 40ng/ml). patients with high concentrations of serum basic protein (0,
Brostoff, S. W., Reuter, W., Hichens, M. & Eylar, E. H. (1974) J. Biol. Chem. 249,559-567 Cohen, S . R., McKhann, G. M. & Guarnieri, M. (1975) J. Neurochem. 25, 371-376 Cohen, S. R., Herndon, R. M. & McKhann, G. M. (1976) N. Engl. J. Med. 295,1455-1457 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194,495-496 McPherson, T. A. & Carnegie, P. R. (1968) J. Lab. Clin. Med. 72, 824-831 Miles, L. E. M. (1976) Protides Biol. Fluids Proc. Colloq. 24, 695-704 Riekkinen, P. J. & Rinne, U. K. (1968) J. Neurol. Sci. 7 , 97-101 Thomas, D. G. T., Palfreyman, J. W. & Ratcliffe, J. G. (1976) Proc. Eur. SOC.Neurochem. Meet. 1st 98 P
Proteolytic Activity in Cerebrospinal Fluid P. TIMOTHY RICHARDS, M. LOUISE CUZNER and ALAN N. DAVISON Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WC1 N 3BG, U.K.
In the cellular immune response, T-lymphocytes, stimulated by antigen, produce lymphokines which recruit the auxiliary cells of the immune system, the polymorphonuclear cells and macrophages. The presence of phagocytic cells in any inflammatory lesion is marked by increased activity of lysosomal hydrolases, particularly of neutral proteinase in polymorphonuclear cells and of acid proteinase in macrophages (Davison & Cuzner,
1977
1425
570th MEETING, CARDIFF I
0 0
0,
4
5
x Cell count
Fig. I . Rate of digestion of 1251-labelledbasic protein by cerebrospinal-fluid polymorphonuclear Zeucocytes ( 0 )and lymphocytes ( 0 )
1977). Cellular infiltration and perivascular cuffing are features of the multiple-sclerosis lesion and there is a predilection for plaques to be located around the ventricles (Adams, 1977). The suggestion that there are factors in the cerebrospinal fluid that contribute to the demyelinating process is further strengthened by the finding of a moderate pleocytosis in the cerebrospinal fluid of patients with multiple sclerosis (Sandberg-Wollheim, 1975). A sensitive enzyme assay has been developed to quantify proteolytic enzyme activity in small volumes of cerebrospinal fluid at acid and neutral pH. The rate of breakdown of 12sI-labelledbasic protein was monitored by polyacrylamide-gel electrophoresis. This method was more accurate than that of counting a trichloroacetic acid precipitate, as myelin basic protein is converted into high-molecular-weight peptides by proteinases, before the appearance of trichloroacetic acid-soluble products. Cerebrospinal-fluid samples (I-2ml) were centrifuged and both the cellular fraction, concentrated 5-lO-fold, and the supernatant were assayed for proteolytic activity. Significant acid proteinase activity was found in the supernatant (44.5pg of basic protein converted/h per ml), whereas in serum there is no detectable activity, owing to the high concentration of circulating a2-macroglobulin, a broad-spectrum proteinase inhibitor (Barrett & Starkey, 1973). The neutral proteinase activity of the cell fraction of the cerebrospinal fluid in central-nervous-system infections was proportional to the polymorphonuclear cell count, the limit of detection being 1-2 cells/mm3 (Fig. I). Lymphocytes did not contribute significant activity to the cellular neutral proteinase. Acid proteinase activity of the cerebrospinal fluid was increased in acute multiple sclerosis and in central-nervous-system infections. These results, in conjunction with neutral proteinase activity of the cell fraction from the cerebrospinal fluid of acutemultiple-sclerosis patients, can be related to the pathogenesis of the disease.
Adams, C. W. M. (1977) Br. Med. Bull. 33, 15-20 Barrett, A. J. & Starkey, P. M. (1973) Biochem. J . 133, 709-724 Davison, A. N. & Cuzner, M. L. (1977) Br. Med. Bull. 33, 60-66 Sandberg-Wollheim, M . (1975) Acta Neurol. Scand. 52, 167-178
Measurement of the Rate of Incorporation in vivo of Amino Acid into Brain Protein WILLIAM F. COULSON and BERNARD HART Corrrtarrld hstitrrte of Biochemistry, The Middlesex Hospital Medical School, London WIP 7PN, U.K.
Determination of the course of incorporation of labelled amino acids into protein has been used widely to measure the rate of protein synthesis in tiivo. Many studies of the effects of loading young rats with phenylalanine on the rate of brain protein synthesis VOl. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
I
I
I
10
20
50
1 I+ 100 NSB
Serum basic protein (ng/ml) Fig. 2. Assay of serum myelin basic protein
.,
Details of the assay are given in the text. Standards contained human myelin basic protein in either pooled normal human serum or blood-bank plasma. Inhibition of tracer binding by human myelin basic protein; NSB, non-specific tracer binding in the absence of specific antisera. 0,V , Inhibition of tracer binding by serial dilutions of sera from 64ng/ml; V , 40ng/ml). patients with high concentrations of serum basic protein (0,
Brostoff, S. W., Reuter, W., Hichens, M. & Eylar, E. H. (1974) J. Biol. Chem. 249,559-567 Cohen, S . R., McKhann, G. M. & Guarnieri, M. (1975) J. Neurochem. 25, 371-376 Cohen, S. R., Herndon, R. M. & McKhann, G. M. (1976) N. Engl. J. Med. 295,1455-1457 Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194,495-496 McPherson, T. A. & Carnegie, P. R. (1968) J. Lab. Clin. Med. 72, 824-831 Miles, L. E. M. (1976) Protides Biol. Fluids Proc. Colloq. 24, 695-704 Riekkinen, P. J. & Rinne, U. K. (1968) J. Neurol. Sci. 7 , 97-101 Thomas, D. G. T., Palfreyman, J. W. & Ratcliffe, J. G. (1976) Proc. Eur. SOC.Neurochem. Meet. 1st 98 P
Proteolytic Activity in Cerebrospinal Fluid P. TIMOTHY RICHARDS, M. LOUISE CUZNER and ALAN N. DAVISON Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WC1 N 3BG, U.K.
In the cellular immune response, T-lymphocytes, stimulated by antigen, produce lymphokines which recruit the auxiliary cells of the immune system, the polymorphonuclear cells and macrophages. The presence of phagocytic cells in any inflammatory lesion is marked by increased activity of lysosomal hydrolases, particularly of neutral proteinase in polymorphonuclear cells and of acid proteinase in macrophages (Davison & Cuzner,
1977
1425
570th MEETING, CARDIFF I
0 0
0,
4
5
x Cell count
Fig. I . Rate of digestion of 1251-labelledbasic protein by cerebrospinal-fluid polymorphonuclear Zeucocytes ( 0 )and lymphocytes ( 0 )
1977). Cellular infiltration and perivascular cuffing are features of the multiple-sclerosis lesion and there is a predilection for plaques to be located around the ventricles (Adams, 1977). The suggestion that there are factors in the cerebrospinal fluid that contribute to the demyelinating process is further strengthened by the finding of a moderate pleocytosis in the cerebrospinal fluid of patients with multiple sclerosis (Sandberg-Wollheim, 1975). A sensitive enzyme assay has been developed to quantify proteolytic enzyme activity in small volumes of cerebrospinal fluid at acid and neutral pH. The rate of breakdown of 12sI-labelledbasic protein was monitored by polyacrylamide-gel electrophoresis. This method was more accurate than that of counting a trichloroacetic acid precipitate, as myelin basic protein is converted into high-molecular-weight peptides by proteinases, before the appearance of trichloroacetic acid-soluble products. Cerebrospinal-fluid samples (I-2ml) were centrifuged and both the cellular fraction, concentrated 5-lO-fold, and the supernatant were assayed for proteolytic activity. Significant acid proteinase activity was found in the supernatant (44.5pg of basic protein converted/h per ml), whereas in serum there is no detectable activity, owing to the high concentration of circulating a2-macroglobulin, a broad-spectrum proteinase inhibitor (Barrett & Starkey, 1973). The neutral proteinase activity of the cell fraction of the cerebrospinal fluid in central-nervous-system infections was proportional to the polymorphonuclear cell count, the limit of detection being 1-2 cells/mm3 (Fig. I). Lymphocytes did not contribute significant activity to the cellular neutral proteinase. Acid proteinase activity of the cerebrospinal fluid was increased in acute multiple sclerosis and in central-nervous-system infections. These results, in conjunction with neutral proteinase activity of the cell fraction from the cerebrospinal fluid of acutemultiple-sclerosis patients, can be related to the pathogenesis of the disease.
Adams, C. W. M. (1977) Br. Med. Bull. 33, 15-20 Barrett, A. J. & Starkey, P. M. (1973) Biochem. J . 133, 709-724 Davison, A. N. & Cuzner, M. L. (1977) Br. Med. Bull. 33, 60-66 Sandberg-Wollheim, M . (1975) Acta Neurol. Scand. 52, 167-178
Measurement of the Rate of Incorporation in vivo of Amino Acid into Brain Protein WILLIAM F. COULSON and BERNARD HART Corrrtarrld hstitrrte of Biochemistry, The Middlesex Hospital Medical School, London WIP 7PN, U.K.
Determination of the course of incorporation of labelled amino acids into protein has been used widely to measure the rate of protein synthesis in tiivo. Many studies of the effects of loading young rats with phenylalanine on the rate of brain protein synthesis VOl. 5
