Neurochemical Research, VoL 20, No. 2, 1995, pp. 137-142 Current Triton X-100 Treatments Do Not Allow a Comp Plasminogen Activator Extraction from Developing Nervo Tissue Susana Pereyra-Alfonso, 1 Gabriel Scieolone, 1 Sara Fiszer de Plazas, ~ Jorge Pecci Saavedra, 1 and Vladimir Flores ~,~ (Accepted September 27, 1994) Determinations of plaminogen activator (PA) activity are usually performed in Triton X-100-treate tissue homogenates or crude membrane fractions. Such preparations usually involve a single Tr X-100 treatment. In the present paper we describe the pattern of variability of PA activity measu in different fractions obtained from the developing chick CNS by a repetitive procedure of Triton X-100 treatment and ultracentrifugation. To further characterize this PA activity we have also performed zymographic analyses during the embryonic development and the early postnatal life. Our results show that: a) a single Triton X-100 treatment does not completely extract the enz and this lead to an underestimation of the total PA activity; b) the PA activity is associated wi the particulate component of the total tissue homogenate requiring its complete solubilization more drastic Triton X-100 treatments; c) better estimations of total and specific activities are obtained by using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions; d) the developing chick optic lobe expresses only one kind of PA molecule along the entire development; e) the level of PA activity vary characteristically during the ontogeny and the early postnatal life indicating the existence of a developmentally regulated mechanism of PA expression. KEY WORDS: Plasminogen activator; central nervous system; optic lobe; chick embryo. INTRODUCTION Determinations of plasminogen activator (PA) ac- tivity in adult (I,2) or developing (3,4) normal tissues or solid rumors (5,6) are usually performed in Triton X- 100-treated(T-treated) tissue homogenates (THs) or 1 Institute of Cell Biology and Neurosciences "Prof. Eduardo De Rob- ertis", School of Medicine, University of Buenos Aires, Paraguay 2155, 2 ~ Piso, 1121-Buenos Aires, Argentina. 2 University Institute of Biomedical Sciences, Favaloro Foundation. Solis 453.1078-Buenos Aires, Argentina. 3 Address reprint requests to: Vladimir Flores, Instituto de Biologia Celular y Neurociencias "Prof. Eduardo De Robertis", Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 2 ~ Piso. 1121-Buenos Aires, Argentina. Telephone number: 01-961-5010 137 crude membrane fractions (CMFs). Values of PA activ- ity obtained under such experimental conditions, which involve a single Triton X-100 treatment, are generally , considered as the total enzyme activity of the tissue and, expressed in terms of specific activity, are usually used for comparisons between different tissues or tumors. As far as we know there are no previous reports in the lit- erature indicating which percentage of the total protease activity is measured under such conditions and there is no a priori validity for the assumption that all the en- zyme activity is actually measured. The aim of this work is to demonstrate that a single Triton X-100 treatment does not completely extract the enzyme from the tissue and thislead to an underesti- mation of the total PA activity. Thus, a correction factor 0364-3190/95/0200-0137507.50/0 9 1995 Plenum Publishing Corporation