A double sandwich enzyme-linked immunosorbent assay (ELISA)
was developed to quantify the proteinaceous inhibitor of
tissue plasminogen activator (t-PA) which occur in the
tissue of Erythrina caffra Thunb. Using the ELISA the t-PA
inhibitor could be detected in nanogramme quantities on the
micro titer plate.
The concentration of the t-PA inhibitor was determined in
different tissues of Erythrina caffra. t-PA inhibitor
concentrations in the order of 1 000 microgrammes per gramme
protein were found in the seeds. Relatively small quantities
of t - PA inhibitor, in the order of 10 to 50 microgrammes
per gramme protein, occurred in root, shoot, leaf and
living bark material.
The t-PA inhibitor was found to accumulate in a similar way
to the storage proteins in developing seeds. The
accumulation of the inhibitor is at a relatively low level
during the early period of seed development but increases
exponentially just before the seeds reach their maximum
size.
The t-PA inhibitor content of the cotyledons decreased
drastically during the process of germination and subsequent
seedling development. The disappearance of the inhibitor
be the result of total degradation of the molecule
can
or partial proteolysis with the modified molecule still being
present in the tissue.
An attempt was made to increase the t-PA inhibitor
content of excised leaves of Erythrina caffra with protein
inducing substances such as polyamines, precursors of
ethylene and phytic acid. The protein inducing compounds
included cell wall hydrolysates of Erythrina caffra, the
marine alga Ecklonia maxima Osbeck (Papenfuss) as well as
Lycopersicon esculentum Mill which induced the, synthesis
of proteinase inhibitors suggested to be involved in the
defense mechanism of plants. None of the substances used,
increased the t-PA inhibitor content of excised leaves or
in vitro cultures of Erythrina caffra. It is suggested that
the t-PA inhibitor is probably not involved in a defense
mechanism of Erythrina caffra.
A callus and suspension culture derived from shoot tissue
was developed to determine the occurrence of the t-PA
inhibitor in vitro. The optimal nutrient medium for the
growth of callus was the salts and vitamins of MURASHIGE and
SKOOG (1962). The medium was supplemented with 3 % sucrose,
0. 1 gramme per litre meso - inositol, 10 micromoles per litre
benzyl adenine and 5 micromoles per litre 2,4-
dichlorophenoxyacetic acid . Different auxins and cytokinins
had a similar growth stimulatory effect on the growth of
callus derived from a number of organs of Erythrina caffra.
The callus from different organs did however, grow at
different rates on the same nutrient medium. Callus derived from leaf, shoot, and cotyledonary tissue grew at similar
rates on the nutrient media of MURASHIGE and SKOOG (1962),
SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and
OJIMA, 1968) despite large differences in the concentration
of the nutrients in the three nutri.ent media. The source of
nitrogen and ratio of nitrate to ammonium was critical to
the growth of callus cultures . The optimal concentration of
nitrate and ammonium was 30 millimoles per litre . The
growth of callus from different organs was significantly
affected by the concentration of sucrose in the nutrient medium.
A concentration of 3% was optimal for callus growth.
Temperature had a significant effect on the growth of
callus. The optimal temperature for callus growth was 25 °C.
A shoot cell suspension culture was established and
maintained at the same temperature and on the same medium
as the callus cultures but with a ten times lower
concentration of growth regulators. A low shake speed was
essential for the growth of the suspension culture. Maximum
growth was obtained at a shake speed of 60 rpm.
Relatively low quantities of t-PA inhibitor, in the order
of 1 to 5 microgrammes per gramme protein, was detected in
the suspension cultures. An attempt was made to increase the
t-PA inhibitor content of the suspension cultures with the
pro te in i nduc i ng compounds used on excised leaves, but
without success. However, the t-PA inhibitor content of the
suspension culture was significantly increased with a ten
times increase in the sulphate content of the nutrient
medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10503 |
| Date | 18 March 2014 |
| Creators | Meyer, Hendrik Johannes. |
| Contributors | Van Staden, Johannes. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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