6~
Medical Research Society
28/67 patients (41%) by PHA tests. There was no significant difference in these tests between the Hodgkin’s and
the non-Hodgkin’s lymphoma groups with the exception
that depressed PHA responses were seen more frequently
in the reticulum cell lymphoma patients, often due to
inhibitory plasma effects. Significantly, more impaired
responses in all tests were seen in patients with disseminated disease. Of sixty-seven patients only seven (all with
disseminated disease) showed depressed responses in all
three tests. E-rosette values were significantly lower in
the Hodgkin’s lymphoma than in the non-Hodgkin’s
lymphoma group. All serum immunoglobulin classes were
elevated in the Hodgkin’s lymphoma group. In lymphocytic lymphoma IgA and IgM levels were depressed.
Neutrophil function was normal or enhanced in all
patients, enhancement being most commonly seen with
disseminated disease.
We conclude that it is important to use several parameters when assessing immunity. Only patients with disseminated lymphoma have profound cellular immunodepression before treatment. We are now investigating the
additional effects of splenectomy and radio or chemotherapy on immune function in our patients.
15. SUBCELLULAR LOCALIZATION OF ENZYMES IMPLICATED IN THE BACTERICIDAL
ACI’IVITES OF HUMAN POLYMORPHONUCLEAR LEUCOCYTES
A. W. %GAL and T. J. PETERS
Department of Medicine, Royal Postgraduate Medical
School, London
(Introduced by Dr G. NEALE)
Microbial killing is thought to be mediated by theenzymic
generation of superoxide and hydrogen peroxide.
Accurate knowledge of the localization of these enzymes
is a prerequisite for an understanding of leucocyte funci
tion. Using the Beaufay automatic zonal rotor, in conjunction with newly developed microassay techniques, we
have determined the subcellular distribution of enzymes
implicated in the generation of superoxide and hydrogen
peroxide.
Highly purified granulocytes were isolated from
peripheral blood and, after homogenization, the 6000 g
min supernatant subjected to isopycnic analytical fractionation on sucrose density gradients (Kane & Peters,
1975, Clinical Science and Molecular Medicine, 49, 171).
The distributions of enzymes with hitherto unknown
localizations were compared with marker enzymes for the
separated organelles.
Plasma membrane, characterized by its content of
5‘ nucleotidase, was shown for the f k t time to contain
a cyanide-insensitive NADPHcytochrome clnitroblue
tetrazolium reductase. This latter activity was inhibited by
superoxide dismutase. Additional reductase activity was
also demonstrated in the mitochondrial-microsomal
fractions.
The primary (azurophil) granules were found to contain NADPH oxidases when assayed in the presence of
MnZ+ and dichlorophenol by the fluorimetric detection
of released NADPH (Lowry et al., 1957, Journal of Bio-
logical Chemistry, 224, 1047). Myeloperoxidase, the
marker enzyme for these granules, is probably responsible
for this oxidase activity (Takanaka & O’Brien, 1975,
Biochemical and Biophysical Research Communlcations,
62,966) and may play only a secondary microbicidal role.
Cytosol fractions, characterized by the distribution of
lactate dehydrogenase, were found to contain superoxide
dismutase, glutathione reductase, glutathione peroxidase
and catalase.
It is suggested that phagocytic vacuoles, formed by the
invagination of plasma membrane, contains a specific
superoxidegenerating NADPH oxidase that has a
bactericidal action. The cytosol contains peroxide and
free radical detoxifying enzymes protecting other cell
components from the damaging effects of these agents.
Subsequent fusion of the phagocytic vacuole with the
primary and secondary granules (Bainton, 1973, Journalof
Cell Biology, 58, 249) permits degradation of the killed
bacteria.
This work is supported by the Medical Research Council and The Wellcome Trust.
16. ROLE OF SPECTRIN I N RED CELL MEMBRANE FUNCTION
S. A. ALI, H. S. S e m and E. C. GORDON-SMITH
Department of Haematology, Royal Postgraduate Medical
School, London
Red blood cell (RBC) shape changes have been correlated
to intracellular ATP content (Nakao, 1974, Cellular and
Molecular Biology of Erythrocytes, p. 35, University Park
Press, Baltimore). A high mol. wt. protein, spectrin,
located at the inner surface of the RBC and comprising
approximately 30% by weight of the total membrane
protein, is thought to be involved in the process of shape
changes. Spectrin can be phosphorylated in the presence
of a CAMP dependent protein kinase using ATP as the
substrate. The protein and the kinase can be partially
pursed and shown to be active in the absence of any
membrane structure. The extracted complex appears to
have a (Caz++ Mg*+)-dependent ATPase activity.
Whether the Caz+-ATPase activity is part of the property
of spectrin-complex or due to some other functional
enzyme is being investigated. Using iodine125 labelling
of the RBC ghost membrane in the phosphorylated and
non-phosphorylated state, it has been possible to detect
conformation changes of the spectrin in the two states. It
is thought that such changes in protein conformation are
not only important in regulation of membrane shape but
may regulate cell life-span and permeability properties of
the cell membrane.
17. LIPID PEROXIDATION O F HUMAN PLATELETS: AN ASSESSMENT OF ITS VALUE IN MEASURING PLATELET LIFE SPAN AND DYSFUNCTION I N MYELOPROLIFERATIVE DISORDERS
J. WHARTON
and J. P. KEENAN
Department of Haematology, University of Liverpool
(Introduced by Professor A. J. BELLINGHAM)
Stuart, Murphy & Oski (1975, New England Journal of
Medicine, 292, 1310) have suggested that inhibition by