Earlier results from other laboratories have shown that beef liver catalase is built up of subunits. However there was no integral relationship between the number of subunits and the number of active sites (heme groups). To clarify the question how the heme groups are distributed among the subunits, the quaternary structure of catalase was studied under different denaturing conditions. 1. 5 M guanidine-HC1, in the presence of 0.1 M @-mercaptoethanol, caused dissociation of catalase into subunits each with a molecular weight of 59,000 ( s ; , ,~ = 2.10 S ; D&o,w = 3.20 F). I n the absence of j3-mercaptoethanol the molecular weight in 5 M guanidine-HC1 was found to be about 140,000 (s;,,~ = 3.50; D;o,w = 2.17 F).2. Succinylation of native catalase with succinic anhydride at neutral pH yielded subunits each with a molecular weight of 65,000 ( S & O ,~ = 3.22 S; D&, = 4.43 F).3. After short incubation (up to six hours) at pH 12.5-12.8 catalase dissociated into subunits each with a sedimentation coefficient ( s &~) of 2.89 s. The molecular weight of these subunits was estimated to be 54,000 and 67,000 respectively. The first value was obtained by combining sedimentation and viscosity measurements ([q] = 17.78 ml/g) from the relation deduced by Scheraga and Mandelkern using a @-value of 2.5 x lo6. The principle of Archibald was used for the estimation of the second value.Longer incubation (e.g. twenty hours) caused splitting into several components, the main component after sixty hours possesses a sedimentation coefficient of 1.7 S. Probably this splitting is due to hydrolysis of peptide bonds. For this reason it was not possible to estimate the diffusion coefficient of the Sz.g-component which needs a dialyzed solution. 4. During the incubation of native catalase in the presence of 7.3-14.6 mM sodium dodecyl sulfate an incomplete dissociation into a component with Treatment of catalase or apocatalase with formic acid followed by incubation with sodium dodecyl sulfate caused dissociation into subunits with a sedimentation coefficient of 3.8 S (D&w = 5.1 F) and 4 S respectively. The molecular weight of the Ss,s-component was found to be 60,000.5. 8 M urea resulted in a dissociation into subunits each with a molecular weight of 133,000
At the present time, attempts are being made in many laboratories to adapt histochemical methods for electron microscopy (1-4, 6, 8, 13, 15, 16, 20-22) and thus localize in greater detail sites of enzymatic activity within the cell, and Barrnett and Palade (5) have recently discussed the problems involved.We asked ourselves whether the well known reaction of Gomori and Takamatsu might be suitable for the demonstration of alkaline phosphatase in the electron microscope. For our experiments we derived valuable suggestions from the investigation of Essner, Novikoff, and Masek (7) on adenosintriphosphatase and 5-nucleotidase localization in liver cells which were recently published in this Journal. As demonstrated by these authors, enzyme inactivation by osmium tetroxide fixation shows a gradient from the periphery to the center of tissue blocks. Applying their technique of prefixation, but for a shorter time (about 3 minutes) we carried out the Gomori reaction (9) at pH 9.6 in the epithelial cells of the kidney tubules of mice. At the end of the incubation period we changed the method of Essner et al. and used a postfixation of 2 hours in osmium tetroxide solution as employed by Caulfield (11).The product of the enzymatic reaction, i.e., calcium phosphate, is absent in the outermost layers of the tissue because enzymatic activity is totally lost at this level. But the activity increases towards the center of the tissue-block. Fixation being conversely best for our purposes at the periphery and worst in the center, it follows that only a small layer, which is wide enough for 20 to 30 sections, shows both the reaction product and a well preserved structure.Calcium phosphate deposits show only little contrast in the electron microscopic image because of their small electron-scattering power. To obtain a better contrast we replaced calcium chloride by lead nitrate in the incubation mixture. But here we ran into several difficulties for most phosphatase substrates give precipitates * The results of this investigation have been presented at the Ninth Meeting of the German Society for Electron Microscopy, held October 19--21, 1959 in Freiburg i. Br. Fuller presentation will appear in the Zeitschrift fiir Histochemie.
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