Abstract
A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.
Highlights
Immunomodulating activity in vivo has been isolated In this study, we cultured G.lucidium mycelia and purified a fromthe mycelial extract of anOriental medicinal novel protein, which we named LZ-8,’from the mycelial fungus, Ling Zhi (Ganoderma lucidium).This protein extract using a bioassay measuring blast-formation stimulawas named Ling Zhi-8 (LZ-8)and itsbiochemical and tory activity toward mouse spleen cells
LZ-8 has an isoelectric point culture medium consisting of 0.24 g of potato dextrose broth and 0.1 g of agar in 10 ml of H20, pH5.7
The mycelia were inoculated onto the slant and cultured for 7 days at 28 “C. In the second stage, the myceliagrown on the slant were transferred into 200 ml of2.4% potato dextrose broth, pH 5.7, contained in a 500-ml flask, and this of 4.4, and sugar analysis indicaateldow carbohydrate suspension was shaken at 110 cpm at 28 “C for 14 days
Summary
Culture of G. lucidium and Purification ofLZ-8"The effect of variation of several culture conditions on mycelia growth was examined. The active fraction was subjected to DEAE-Sephadex A-25 ion-exchange chromatography, with a typical elution profile revealing five majorpeaks of 280 nm absorbing material (Fig. 1B). A single band was observed using the recently described Tricine-SDS-polyacrylamide gel electrophoresis (lo), but an apparent M , = 13,100 of LZ-8 was obtained in the presence of 2-mercaptoethanol (Fig. 2, lane 6 ). No aggregation was observed between any types of human red fraction).Peakfractions (A-E) were obtained by pooling column blood cells in the presence of purified LZ-8 within the con-. LZ-8 activity was determined by two methods, hemagglutination assay using sheep red blood cells and blast-formation stimulatory activity using mouse spleen lymphocytes (values in parentheses), as described.
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