LINDERSTR0M-LANG ET AL 



179 



LINDERSTR0M-LANG ET AL 



Studies in Enzymatic Histochemistry 

 soon had contributions from T. 

 Philipson, who investigated the 

 peptidase activity of single centri- 

 fugally separated parts of eggs of 

 Psammechinus miliaris {Ibid, 1934, 20, 

 No. 4), and D. Glick, who elaborated 

 an acidimetric method for lipolytic en- 

 zymes {Ibid, No. 5) and applied it to a 

 study of the distribution of esterase in 

 the gastric and duodenal mucosa of the 

 hog (Glick, D. 76id, No. 11). 



The latter was undertaken since Lin- 

 derstr0m-Lang and Holter had already 

 underway similar investigations of the 

 distribution of pepsin {Ibid, 1935, 20, 

 No. 11), acid {Ibid, 20, No. 11), and 

 peptidase {Ibid, 20, No. 11). A. S0e- 

 borg-Ohlsen joined in a collaborative 

 study of the enzj'me distribution in the 

 hog stomach as a function of its histo- 

 logical structure {Ibid, 20, No. 11) and 

 the enzyme pattern in the mucosa was 

 revealed to a significant degree. 



The need of methods for the chemical 

 determination of additional cellular 

 constituents diverted the Carlsberg 

 Laboratory workers and visiting scien- 

 tists to the task of elaborating them, 

 and for the visitors this served the ad- 

 ditional purpose of providing excellent 

 first hand training and experience in 

 the use of the techniques. With 

 Palmer, A. H. {Ibid, 1935, 21, No. 1), 

 an electrometric chloride titration was 

 developed with a precision of about 

 14 X 10-' g. of chloride; Levy, M. {Ibid, 

 1936, 21, 101-110) refined the Kjeldahl 

 nitrogen analysis to 3 X 10""® g. of 

 nitrogen, Linderstr0m-Lang (Compt. 

 rend. trav. lab. Carlsberg, Ser. Chim., 

 1936, 21, 111-122) worked out an electro- 

 metric titration method for sodium plus 

 potassium sensitive to 1 X 10"^ m. 

 equiv., Norberg, B. {Ibid, 1937, 21, 

 233-241) evolved a titrimetric estima- 

 tion of potassium isolated as the 

 iodoplatinate that was accurate to 1-2 

 X 10— ' m. equiv., and with Weil, L. 

 {Ibid, 1935, 21, 7-14) micro methods for 

 arginase and urease were established. 



Linderstr0m-Lang, K. and Engel, C. 

 {Ibid, 1938, 21, 243-258) employed the 

 method that had been previously de- 

 veloped for reducing sugars for the 

 measurement of amylase activity, the 

 distribution of which they studied in 

 barley, and Holter and Doyle, W. L. 

 {Ibid, 1938, 22, 219-225) subsequently 

 modified the technique to gain greater 

 precision. Meanwhile micro methods 

 for other enzymes were being developed. 

 Holter and Doyle (J. Cell Compt. 

 Physiol., 1935, 12, 295-308) adapted the 

 iodometric method of Stern to the esti- 

 mation of catalase with a precision 



equivalent to the decomposition of 2 X 

 10"* g. of hydrogen peroxide, and Glick, 

 D. (Compt. rend. trav. lab. Carlsberg, 

 Ser. Chim., 1938, 21, 2G3-268) extended 

 his esterase method to include the meas- 

 urement of cholinesteraso with a sensi- 

 tivity equivalent to the hydrolysis of 

 1 X 10~* mole of ester. 



As the new methods were made avail- 

 able their application to specific prob- 

 lems followed at once. Thus, Linder- 

 str0m-Lang, K., and Duspiva, F., (Ibid, 

 1936, 21, 53-84) studied the digestion of 

 keratin by larvae of the clothes moth, 

 and Duspiva followed this work by in- 

 vestigations of various proteolytic en- 

 zymes of clothes- and wax-moth larvae 

 {Ibid, 1936, 21, 177-202), as well as of 

 pH of the intestinal juice of these organ- 

 isms for which a micro glass electrode 

 was used {Ibid, 21, 167-176). Holter (J. 

 CellComp. Physiol., 1936, 8, 179-200) 

 studied peptidase localization in marine 

 ova, with Kopac {Ibid, 1937, 10, 423- 

 437) the localization of this enzyme 

 in amoeba, and with Doyle, W. L. 

 (Compt. rend. trav. lab. Carlsberg, Ser. 

 Chim., 1938, 22, 219-225) the amylase 

 localization in amoeba. Doyle {Ibid, 

 1938, 21, 291-299) also investigated the 

 catalase and peptidase activity in ma- 

 rine ova. The activation of leucylpep- 

 tidase of single Tubifcx eggs by magnes- 

 ium salts was reported by Holter, H., 

 Lehmann-Bern, F. E. and Linderstr0m- 

 Lang (Compt. rend. trav. lab. Carls- 

 berg, Ser. Chim., 1938, 21, 259-262). 



Other applications in this period in- 

 clude a study of the distribution of 

 urease in the dog stomach by Linder- 

 str0m-Lang and S0eborg-Ohlsen, A. 

 (Enzymologia, 1936, 1, 92-95), and an 

 investigation of the cholinesterase dis- 

 tribution in the hog stomach before and 

 after administration of drugs that affect 

 gastric secretion by Glick {Ibid, 1938, 

 21, 269-281). Using a titrimetric dye 

 method previously worked out (J. Biol. 

 Chem., 1935, 109, 433-436) Glick fol- 

 lowed the changes in ascorbic acid in 

 different parts of the developing barley 

 embryo up to the ten-day sprout stage 

 {Ibid, 1937, 21, 203-209). 



Titrimetric procedures alone had 

 been exploited up to this time. But in 

 the attempt to extend the applicability 

 of this approach to quantitative histo- 

 chemistry, other analytical principles 

 were tested. Colorimetry, if conducted 

 on the required micro scale, would ob- 

 viously open a vast new region for 

 exploitation, but the limiting factor in 

 the middle nineteen thirties was the 

 availability of the necessary equipment. 

 At that time, the Zeiss Pulfrich step 

 photometer, that employed cuvettes re- 



