I956 the mixing chamber before the reagents are emptied into the reaction vessel. The accuracy of mixing exceeds the limit of resolution of the spectrophotometer. 3. The reaction vessel consists of a cylindrical quartz tube held in position with unstretched nylon bushings in a brass block adjacent to the cuvette housing unit. The flow of material is controlled by a needle valve at the exit of the reaction-vessel housing unit. Approximately 6 ml. of fluid is required to flush out the reaction vessel. 4. Temperature control is maintained with a water jacket round the four syringes and with cooling channels within the reaction-vessel housing unit. Steady-state temperature is reached in the syringes in a maximum of 6 min. 5. The signal from the density-scale slide-wire potentiometer of the spectrophotometer is fed through a potential divider into a Brown stripchart recorder. As the operator tracks the changes in optical density of the reaction mixture the percentage transmission is recorded continuously. By means of a mixing switch the temperature of the reaction vessel measured with a thermopile is recorded at intervals during the reaction time. 6. The percentage error (standard deviation) of the velocity constant of decomposition of hydrogen peroxide by bacterial catalase measured with this apparatus is less than ± 2 %.

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1. Crude extracts of Aspergillus oryzae grown under conditions of sulphur limitation possess high arylsulphatase activity. 2. This activity can be greatly enhanced by the inclusion of tyramine or a number of other phenols in the assay medium. 3. The arylsulphatase activity of these extracts can be resolved into three distinct fractions by chromatography on DEAE-cellulose. 4. The effect of tyramine is restricted to one of these fractions only. 5. Evidence is presented which indicates that this effect is the consequence of a phenol sulphotransferase activity, which shows no requirement for 3'-phosphoadenosine 5'-phosphate as a cofactor, and which will not transfer sulphate from 3'-phosphoadenosine 5'-sulphatophosphate to potential phenolic acceptors. 6. The three enzymes differ also in their molecular weights and substrate specificities.

1. Homogenates of Chinese hamster fibroblasts have been fractionated by analytical centrifugation techniques. Lysosomes have been characterised and identified by the distinctive behaviour of specific marker constituents.2. The acid hydrolases of hamster fibroblasts were located in two distinct organelles, characterised by differences in their equilibrium densities and rates of sedimentation. The lighter population of lysosomes with a modal density of 1.10 g cm-3, contained glycosidases, nucleases and acid phosphatase. The denser population, with a modal density of 1.15 g ~r n -~, contained proteases and arylsulphatase B.3. Acid hydrolases in both populations showed similar degrees of latency and structure-linked sedimentability . The heterophagic activity of hamster fibroblasts was investigated by growing the cells in the presence of ['4C]sucrose, ['4C]inulin and Triton WR 1339. These compounds are taken up by the cells and appear to be located in the denser population of lysosomes.5. The low density of the lighter population of lysosomes appears to result from a high content of cholesterol esters and triacylglycerols. It is suggested that the lysosomes may be the site of storage of a considerable proportion of these lipids in cultured hamster cells.The increased use of cultured mammalian cells in the study of lysosomes and in many other areas of subcellular organelle research has necessitated the characterisation of organelles in such cells. Cultured cells are especially suited to this type of study since ambiguities arising from the presence of more than one cell type are minimised and the effects of compounds which interact with subcellular components can be investigated under precisely defined conditions. Cell lines which have been investigated by subcellular fractionation techniques include rat embryo fibroblasts Preliminary observations on hamster ovary fibroblasts [2] suggested that, despite the use of a homogeneous cell population, acid hydrolases were located within two distinct subcellular organelles characterised by differences in their equilibrium densities. In the present study, the lysosomes of Chinese hamster CH-23 fibroblasts [4] (a cell line similar to the ovary

1. A method is described for following continuously the action of beta-galactosidase on 4-methylumbelliferyl beta-D-galactoside at pH 4.5, in which 4-methylumbelliferone production is measured at fluorescence excitation and emission wavelengths of 324 and 444nm respectively. 2. Initial-rate studies show that the presence of salt activates beta-galactosidase up to 100 mM, but is inhibitory above that concentration. The enzyme is very unstable at 37 degrees C and low ionic strength, but stability increases with ionic strength. 3. The stability of the enzyme at 37 degrees C decreases markedly with rising pH in the range 5.9--8.0. 4. Gel-filtration patterns demonstrate that there is a marked tendency to polymerization with increasing ionic strength. The gel-filtration pattern shows decreasing amounts of dimer with increasing pH. 5. The correlation between activity, stability and molecular form of beta-galactosidase is discussed. It is suggested that the dimeric form of the enzyme is the most stable and active form. The implications of this finding for the assay of beta-galactosidase under physiological conditions for prenatal diagnosis are discussed. 6. Evidence for the possible occurrence of a 36 000-mol.wt. from of beta-galactosidase is presented. 7. A computer program for the calculation of initial rates has been deposited as Supplementary Publication SUP 50114 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.