Spleen extraction technique

This same extraction technique can be applied to erythrocytes, neutrophils, and other comparable cells. A similar approach also can be used with tissues (such as liver and spleen) employing 7-8 ml chloroform-methanol-water per gram of homogenized tissue. In the latter case, the extraction period should be a minimum of one hour at room temperature. 

Nucleotidase present in 48,000 X Q supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (7-4). 5 -UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75). This enzyme hydrolyzes all ribo-and deoxyribonucleoside 5 -phosphates (except dCMP and dTMP) with preference for 5 -IMP and 5 -UMP. The enzyme is strongly activated by Mn2+ Mg2+ is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (88, 86). 5 -Nucleotidase in mammary gland hydrolyzes all 5 -ribonucleotides and shows a decrease from pregnancy to early lactation (76). Rats injected with glucagon show increased 5 -nucleotidase in pancreatic islet tissue (77). The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isozymes (75). Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79). 

Another group of experimentors tried to study the preferential utilisation of intact proteins or partial hydrolysates by means of the metabolic trap technique, which consists in measuring the effectiveness with which a labeled free amino acid can compete with larger protein fragments in the formation of new protein. The most positive evidence for the direct utilisation of such fn ments was generally found with embryonic and tumor tissues. Thus Ebert (343) found that when transplants of chick embryo kidney, liver, or spleen, labeled with S -methionine, were made into chick embryos, the radioactivity appeared predominantly in the correspondit organ, and that this transfer of radioactivity could not be inhibited by free methionine. Similarly, Francis and Winnick (344) found that the soluble proteins of a chick embryo extract were utilized in preference to free amino acids by embryonic heart cultures and that, furthermore, p-fluorophenyl-alanine did not inhibit the transfer of phenylalanine from the embryo extract proteins. These experiments, however, are open to many interpretations, especially since functional adoption (or merely adsorption) of the exogenous proteins by the different tissues has not been excluded. 

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