A high resolution technique for the fine‐structural localization of acid hydrolases

Abstract

Summary An azo dye technique is described for the fine‐structural localization of acid hydrolases. Enzymatically released primary reaction product, an insoluble substituted naphthol, is coupled with ρ‐nitrobenzene diazonium‐tetrafluoborate to produce a dense particulate azo dye. Simultaneous and post‐coupling procedures are employed. The method is potentially applicable to all acid hydrolases and is free from the artifacts which beset the commonly used lead salt methods. The localization of β‐glucuronidase and acid phosphatase in rat and slug liver is specifically dealt with here. Lysosomal and cytoplasmic sources of enzyme activity are revealed.

Sites of acid phosphatase in the differentiating root protophloem of Nymphoides peltata (S.G. Gmel.) O. Kuntze.

“…are the only previous authors to have investigated ultrastructurally the position of acidphosphatase activity in sieve elements using an azo-dye method. These authors used the azo-dye method of Bowen (1971). We also attempted this method with our material but found the azo-dye to be soluble in acetone.…”

The use of X‐ray microanalysis to investigate problems encountered in enzyme cytochemistry*

“…Due to the inherent faults of the lead salt methods for localizing enzymatic activity at electron microscope level, (Goldfischer, Essner & Novikoff, 1964), there have been several attempts at developing alternative techniques based on azo dye cytochemistry (Lehrer & Ornstein, 1959;Tice & Barrnett, 1965;Bowen, 1968Bowen, , 1971 ;Hayashi, Shirahama, Cohen, 1968;Smith & Fishman, 1968;Livingston et al, 1969). Azo dye methods, however, have certain drawbacks.…”

“…The use of substituted naphthols in azo dye electron cytochemistry has already been established (Hayashi et al, 1968;Smith & Fishman, 1968;Livingston et al, 1969;Bowen, 1971Bowen, , 1973. Enzymatically released Naphthol ASTR, provides an insoluble, substantive primary reaction product, containing a co-valently linked chlorine atom.…”

Acid phosphatase activity in intercellular spaces in roots of Nymphoides peltata (S. G. Gmel.) O. Kuntze

“…The roots were then washed for 1 h in cacodylate buffer and cut into 50 /mi slices on a Mcllwain tissue chopper (Mickle Laboratoty Engineering Co,), The sections were washed overnight in 0,1 kmol m ~ •' cacodylate buffer and incubated the following day at pH 4,8 in lead-salt media containing one of the substrates cytidine 5' tnonophosphate (CMP), sodiutn /?glycerophosphate (/^GP) or p-nitrophenyl phosphate (p-NPP); see Oparka (1979) and Oparka et al (1981). Additional sections to be examined by light microscopy were incubated in Bowen's (1971) After incubation in the appropriate tnediutn the sections were postfixed in 1% osmiutn tetroxide buffered with 0,1 ktnol m"^^ sodium cacodylate (pH 7,2), dehydrated through a graded acetone series and embedded in Epon resin. Material incubated in leadsalt media was cut on glass knives in an LKB ultrotome 111 ultramicrotome, dried on glass slides and stained with !…”

“…'/" atntnonium sulphide solution for 2 tnin. Fifty /an sections incubated in the azo-dye tnedium of Bowen (1971) were viewed directly utider the light tnicroscope after incubation since dehydration in acetone tnay cause a loss of azo dye frotn the tissue (Oparka, 1979), Sections for electron tnicroscopy were cut on glass knives and viewed unstained in a Philips EM 301 electron tnicroscope operating at 60 kV, Control incubation tnedia either lacked substrate or contained 0,01 kmol tn"^ sodiutn fluotide as an enzytne inhibitor, Additiotial matetial for light tnicroscopy was fixed as described above and etnbedded in paraffin wax by standard procedures. Ten /(tn sections were cut on a t-otary micrototne, cleared in xylene and stained with 1% toluidene blue.…”