LYSOSOMES IN LYMPHOID TISSUE I. The Measurement of Hydrolytic Activities in Whole Homogenates

Methods have been developed for the quantitative assay of cytochrome oxidase, esterase, and 11 acid hydrolases in rat-spleen homogenates. These methods seem to be applicable also to other lymphoid tissues. Preliminary studies, extended to nine of the acid hydrolases, indicate that these enzymes occur in partly latent and sedimentable form and that they can be unmasked and rendered soluble by some of the treatments that liberate the enzymes from rat-liver lysosomes. The spleen particles appear to be very sensitive to mechanical injury, a property which necessitates special precautions in homogenizing the tissue. Agglutination of spleen particles takes place to a larger extent in 0.25 M sucrose than in 0.15 M KC1. I N T R O D U C T I O N Lymphoid tissues, especially the spleen, represent one of the richest sources of many of the acid hydrolases known to be associated with lysosomes in other tissues. A lysosomal localization of these enzymes in lymphoid tissues is suggested by the finding that several of them occur partly in latent, particle-bound form, in freshly prepared homogenates of spleen or thymus (6, 10, 22, 26-28, 33). However, various investigators, using different techniques with these tissues, have obtained rather variable patterns for the intracellular distribution of individual acid hydrolases (2, 6, 15, 22, 29, 30) and this fact has even led some workers to question the existence of lysosomes as a distinct group of cytoplasmic particles (22). Our investigations were started with the aim of obtaining more complete and accurate information concerning the existence, properties, and functions of lysosomes in lymphoid tissues. They were performed principally on spleen, but some experiments were also carried out on thymus and on lymph nodes. The approach has been essentially biochemical and has been extended to about a dozen different enzymes in order to provide a basis as broad as possible for further morphological studies. The present paper describes the results of experiments dealing with the measurement and main kinetic properties of enzymes in whole homogenates. The association of these enzymes with lysosomes and various data concerning the cellular location and functional properties of these particles form the subject of subsequent papers in this series. M A T E R I A L S A N D M E T H O D S The experiments werc performed on Sprague-Dawlcy rats of cither sex weighing bctwccn 150 and 300 g. The animals were fed Purina Chow and were permitted access to food and water until the time of the experiment; they were killed by dccapitation. The splccn and other lymphoid tissues wcrc quickly rcmoved, immerscd in a tared beakcr containing ice cold 0.15 M KC1, and weighed. Some dctcrminations for acid hydrolase activity wcre made on cell suspensions obtained from thymus. Cells were teased into Tyrode's solution according to 325 on O cber 0, 2017 jcb.rress.org D ow nladed fom the method described by Fastier (16). The preparation was filtered through several layers of gauze and the cells were collected by centrifuging at 100 g for 10 min. They were then washed two or three times and resuspended to the desired volume. The manner in which the tissue and cell preparations were dispersed and analyzed for various enzymes will be described under Results. The following substrates were purchased from the Sigma Company (St. Louis, Missouri): fl-glycerophosphate (Grade I), cytochrome c (Type III) , DNA (Type I), hemoglobin (Bovine, Type I, 2x crystallized), nitrocatechol sulfate (Dipotassium salt), 0-nitrophenyl acetate (Practical grade), o-nitrophenyl-13-galactoside, phenolphthalein /3-glucuronide (Free acid), and RNA (Commercial grade, yeast). Benzoyl-L-arginine amide hydrochloride and glyeylL-tyrosine amide acetate were obtained from Mann Research Laboratories (New York) and freed from contaminating ammonia by distillation in the presence of K~COz, followed by neutralization of the carbonate, p-Nitrophenyl-a-maunoside was synthesized according to the method of Je rmyn (21), with some additional instructions from Dr. James Conchie. The final product had a melting point of 182°-183°C, as compared with 180°-181°C reported by Conchie et al. (7). p-Nitrophenyl-N-acetyl-~glucosaminide was synthesized by the method of Findlay et al. (17). The melting point was 201°-202°C, as compared to the value of 204°C given by these authors. Protein was measured according to Lowry et al (23).