Robert E. Plapinger

MEMBRANEOUS ULTRASTRUCTURAL DEMONSTRATION OF AMINOPEPTIDASE AND γ-GLUTAMYL TRANSPEPTIDASE ACTIVITIES WITH A NEW DIAZONIUM SALT THAT YIELDS A LIPOPHOBIC, OSMIOPHILIC AZO DYE

When 4-aminophthalhydrazide is freshly diazotized and used as the coupling agent in demonstrating aminopeptidase activity or γ-glutamyl transpeptidase activity, with substrates that yield 4-methoxy-2-naphthylamine or even 2-naphthylamine, azo dyes are produced which are readily osmiophilic. Although the distribution of the reddish azo dyes is similar to that noted with earlier methods in light microscopy, the distribution of the deposits in electron microscopic preparations is distinctly membraneous in location in contrast to the droplet distribution of previous methods. We attribute membraneous localization of enzymatic activity with the new methods presented here to the lipophobic properties of the azo dyes provided by their 4-aminophthalhydrazide (4-APH) moiety. The new agent 4-aminophthalhydrazide was designed with this in mind.

AN OSMIOPHILIC DISTYRYL DITETRAZOLIUM SALT (DS-NBT) FOR ULTRASTRUCTURAL DEHYDROGENASE ACTIVITY

The preparation of a new osmiophilic ditetrazolium salt (DS-NBT) containing two styryl groups, but retaining the essential structural features of nitro-BT, is given. DS-NBT may be used to demonstrate succinic dehydrogenase activity (SDH) faster than with nitro-BT and fully as rapidly as with TC-NBT. DS-NBT is more stable than TC-NBT and it is more reliably and more readily osmicated than TC-NBT. The ultrastructural sites of SDH and NADH2 diaphorase activity are similar to those shown for cytochrome oxidase activity with diaminobenzidine in rat heart. The sites of lactic dehydrogenase activity are found in the sarcoplasmic reticulum and mitochondria of rat skeletal muscle fixed in formaldehyde for 3 hr. Tissue penetration of the doubly charged ditetrazolium salt (DS-NBT) was facilitated by 40-sec sonication and by 5% dimethyl sulfoxide. Illustrative examples and controls are included.

Role of some structural features of subtrates on trypsin activity

Abstract Tryptic hydrolysis of 27 different amides of arginine was studied in an attempt to learn more about the specificity requirements of trypsin. Chain length is important for tryptic action, the optimal length being one which contains four amides (pentalysine) or three amides and one urethane (carbobenzoxy-triarginyl naphthyl amide). The electronic characteristics of the groups blocking the α-amino group of arginine appear to play a role in that the more negatively charged groups are hydrolyzed more readily by trypsin. The properties of the amine attached to the enzyme-sensitive bond also have an influence on tryptic action. Aromatic amides are much more susceptible than simple amides in dipeptide and tripeptide substrates. The three most active substrates, Nα-carbobenzoxydiglycyl-l-arginyl-2-naphthylamine, β-carboxyproprionyl-l-diarginyl-2-naphthylamide, and Nα-carbobenzoxy-l-triarginyl-2-naphthylamide, were found to be split more readily than Nα-benzoyl-l-arginine-2-naphthylamide (BANA) by factors of 220, 147, and 113, respectively. As little as 5 μg of added trypsin per milliliter of serum can be identified in serum. A smaller amount could not be measured in spite of a series of attempts to separate trypsin from the anti-trypsin present in serum.

Some cytochemical correlations between oxidase activity (cytochrome and peroxidase) and chemical structure of bis(phenylenediamines)

SummaryA series of 17 bis(phenylenediamine) derivatives have been prepared and compared with 3,3′-diaminobenzidine (DAB) with regard to their ability to demonstrate cytochrome oxidase activity, peroxisome activity, horseradish peroxidase activity, erthrocytic peroxidase activity in cytochemical preparations, and bovine catalase activity in in vitro experiments. The results are tabulated, some illustrative photomicrographs are included and interesting correlations are discussed.

Preparation and structure-activity relationship of reagents for cytochrome oxidase activity: potential for light and electron microscopy

SummaryThe syntheses of a series of p-substituted aromatic diamines and some representative thioureido- and mercapto-naphthols are described. Their cytochemical behavior in the Nadi reaction using fresh frozen sections of rat heart was studied in an attempt to learn more about the structural requirements of the Nadi reaction for cytochrome oxidase.The lipid solubility of the dyes formed from either N,N-dimethyl-p-phenylenediamine or ADN (4-amino-1-N,N-dimethylnaphthylamine) with 1-naphthol can be decreased by substituting hydrophilic hydroxyethyl or dihydroxypropyl side chains for one or more of the methyl groups in these reagents. However, these dyes diffuse readily, fade rapidly and are too soluble in water as well as lipid, which renders them unsuitable for light and electron microscopy.Burstones reagent PPD (N-phenyl-p-phenylenediamine) and ADN (4-amino-1-N,N-dimethylnaphthylamine) are capable of undergoing self condensation either alone or in the presence of an unreactive naphthol to give colored indamines which osmicate readily. Blocking either the 4-position of N-phenyl-p-phenylenediamine, the secondary amino group, or both of these positions, with a methyl group did not prevent indamine formation.N-Benzyl-, N-4-methylbenzyl- and N-4-methoxybenzyl-p-phenylenediamine are good reagents for the demonstration of cytochrome oxidase activity in the Nadi reaction with light microscopy. They produce blue indoaniline dyes with 1-naphthols and do not self condense to form highly colored pigments. These indoanilines are osmiophilic eliminating the necessity for introducing osmiophilic groups for electron microscopic studies. Their drawback in electron microscopy is in the droplet nature of the deposit.Incorporating a carbonyl or carboxyl function into the 2-position or a bulky substituent into the 5-position of 1-naphthol prevented indoaniline formation.A new method for the cytochemical demonstration of cytochrome oxidase is presented utilizing a new reagent N,N′-bis(p-aminophenyl)-1,3 xylylenediamine (XIII) (BAXD). This reagent is polymerized to an insoluble osmiophilic polymer distributed in non-droplet form, thus providing a useful method for demonstrating cytochrome oxidase activity in light and electron microscopy.

Comparison of the ultrastructural demonstration of cytochrome oxidase activity with three Bis(Phenylenediamines)

SummaryThree bis(phenylenediamines) are compared in formaldehyde-fixed rat liver and rat heart. Diaminobenzidine (DAB) demonstrated cytochrome oxidase on mitochondrial cristae, BAXD demonstrated both mitochondrial cytochrome oxidase and a terminal oxidase in endoplasmic reticulum and sarcoplasmic reticulum and BED demonstrated a terminal oxidase only on endoplasmic reticulum and sarcoplasmic reticulum.

COMPARISON OF ULTRASTRUCTURAL CHOLINESTERASE DEMONSTRATION IN THE MOTOR END PLATE WITH α-ACETYLTHIOL-m-TOLUENEDIAZONIUM ION AND 3-ACETOXY-5-INDOLEDIAZONIUM ION

The first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form an osmiophilic polymer was reported earlier. The first results were obtained with substrates containing an enzyme-susceptible thiolester group and a diazonium group in the same molecule, which yielded osmiophilic polymers upon enzymatic hydrolysis via diazothioether formation linking the units. An enzyme-susceptible ester group has also been incorporated in the same indole molecule with a diazonium group to yield an osmiophilic polymer upon esterolysis via coupling to azo dye formation linking the units. The schemas of these two systems are given. The thiol substrate is α-acetylthiol-m-toluenediazonium ion (ATTD, compound II) and the indoxyl substrate is 3-acetoxy-5-indolediazonium ion (AID, compound VII). The preparation of AID is given here. Because of the strongly positive charges on the diazonium ion, neither ATTD nor AID is hydrolyzed by the aliesterases of rat kidney. The results with ATTD and AID in the ultrastructural demonstration of cholinesterases in the motor end plate of the rat are compared, as well as the influence of varying the method of fixation, substrate concentration, duration and temperature of incubation and the use of selected inhibitors with ATTD. Evidence was provided with ATTD that both acetylcholinesterase and pseudocholinesterase are present on the inner surface of the synaptic clefts of the motor end plate and that the deposits are discrete and not linear as has been reported with some other methods. On the other hand, deposits with AID are more finely granular and are also limited to the inner aspect of the synaptic clefts. The reasons for the differences in appearance of the deposits in ultrastructural preparations are discussed.

LOCALIZATION OF ACETYLCHOLINESTERASE VIA PRODUCTION OF OSMIOPHILIC POLYMERS: NEW BENZENEDIAZONIUM SALTS WITH THIOLACETATE FUNCTIONS

By incorporating an enzyme-susceptible thiolester group and a diazonium group into the same molecule, it has been possible to obtain an osmiophilic polymer upon enzymatic hydrolysis of the thiolester, via diazothioether formation linking the units. The thiolacetates with diazonium groups are specific substrates for cholinesterase because of the strong positive charge on the diazonium group. Following osmication, sites of acetylcholinesterase in motor end plate are notable as black deposits in light microscopy and opaque deposits in electron microscopy. This is the first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form a polymer. The insolubility of the polymer in both water and lipid before osmication provides precision of localization needed for the high resolution of electron microscopy. However, the effectiveness of the new principle for electron microscopy depends as well upon the rate of the capture reaction (polymerization), which in turn depends upon the pK a of the thiol and pH and temperature at which the cytochemical reaction is conducted.

Assay of trypsin activity of human serum with a chromogenic substrate, CBZ-diglycyl-l-arginyl-2-naphthylamide hydrochloride (GGANA)

Abstract Recent development of a very sensitive chromogenic substrate (GGANA) for trypsin and reports of proteins (P) in serum which form inhibitor-resistant active complexes (PT) with trypsin (T) have prompted an investigation of trypsin-like activity of serum. GGANA was sensitive to T and to normal human serum but was not affected noticeably by proteolytic enzymes of serum once considered trypsin-like (that is, thrombin, fibrinolysin, and kallikrein). Though serum inhibited T to some degree, GGANA could be used to measure small quantities of T added to serum (as little as 0.05 μg./ml.). The rate of inhibition of the tryptic activity, however, was inversely proportional to the concentration of the T in the serum. Presumably this phenomenon occurs because the fraction of T that can be bound by the small quantity of high molecular weight protective protein (P) to form the active complex PT in the presence of inhibitors (I) is closer to 100% in a large excess of serum which can provide an effective concentration of P. In the absence of an effective concentration of P some of the T can be bound by I to form inactive IT, PT is irreversibly inactivated by acid while IT apparently gains some activity on acid treatment followed by neutralization. Since normal serum irreversibly loses its activity on acid treatment, it probably contains PT (endogenous). How PT, P, I, and IT activity of serum changes with acute pancreatitis would be difficult to predict. The serum trypsin-like activity of 171 normal individuals averaged 391 Klett units per 30 minutes with an upper limit of 785 and a lower limit of −4. The activity found for four cases of acute pancreatitis averaged −19. The completed study of the variation of PT in serum in pancreatitis using the chromogenic peptide, GGANA, as a substrate will be reported later.

Species-dependent pleomorphic behavior of pancreatic tryptic activity☆

Abstract Homogenates of canine and human pancreas enriched with trypsin of endogenous origin only were obtained when such homogenates were incubated at 0°C. with aliquots of bovine trypsin (Tryptar) rendered insoluble by mechanical entrapment inside the lattices of polyacrylamide gel. Insoluble bovine trypsin was able to activate pancreatic trypsinogen and overcome pancreatic trypsin inhibitor prior to its removal by centrifugation. In addition, it was observed that some human pancreases readily developed tryptic activity in the usually long interval between death and autopsy, and that others generated substantial activity after homogenization and incubation at 0°C. In contrast, homogenates of canine pancreas required the addition of exogenous trypsin for conversion of trypsinogen to trypsin. When the generated tryptic activity of pancreatic homogenates was compared with that of purified bovine trypsin, human trypsin differed from bovine and canine trypsin in its substantially lower inhibition by ovomucoid and soybean trypsin inhibitor. The ease of conversion of human trypsinogen to trypsin, the stability of the latter, its lower sensitivity to trypsin inhibitor, and variation in content of the inhibitor are undoubtedly factors in the etiology of pancreatitis, and they may explain the significant rate of occurrence of this disease in man.