Gerhard Pfleiderer

The evolution of endopeptidases. XII. The proteolytic enzymes of the honeybee (Apis mellifica L.)

Abstract 1. 1. Four fractions (A–D) with endopeptidase activity have been isolated and characterized from the midgut of adult worker honey bees. 2. 2. Fraction A exhibited a strictly basic cleavage specificity when tried on the B chain of oxidized insulin, splitting exactly as bovine trypsin only the bonds Arg 22 /Gly 23 and Lys 29 /Ala 30 . Its molecular weight is 20,000. 3. 3. From the other fractions, B and C do not resemble mammalian pancreatic proteases, but fraction D shows a cleavage specificity similar to chymotrypsin. 4. 4. With the exception of fraction C all other fractions form part of the group of “serine”-proteases. 5. 5. Clear differences were visible when the occurrence of these proteases was studied in adult workers, drones and queens. 6. 6. Apis cerana and Apis mellifica show immunologically cross-reacting material with identical cleavage specificity but different electrophoretic mobility.

The synthesis of spin-label derivatives of NAD+ and its structural components and their binding to lactate dehydrogenase

Spin-labelled derivatives of NAD+ and its structural components (i.e. adenosine, adenine, AMP, ADP and ADPR) have been synthesized. Their binding to pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been studied and dissociation constants have been determined. The spin-labelled derivatives of ADP and ADPR exhibit a tighter binding than the corresponding NAD+ derivative. This may be attributed to the repulsion of the positively charged nicotinamide ring by an histidine side chain in the active center of the enzyme.

The cellular distribution of aldolase isozymes in rat kidney and brain determined in tissue sections by the immuno-histochemical method

SummaryThe use of the immuno-histochemical method permits the localization of aldolase isozymes in tissue sections. Upon incubating a section with a monomer-specific antiserum, isozymes containing that monomer remain in the section, whereas other cytoplasmic enzymes diffuse out of the section. If soluble antigen is added subsequently, it is bound by the tissue-bound antibody. These antibody fixed aldolases can then be stained by the use of a tetrazolium test linked to substrate hydrolysis.In this way it was demonstrated that isozymes of aldolase containing mostly the A monomer are predominantly localized in the distal tubules, the collecting tubules, the vessels and capillaries of the kidney, the ganglia, the Purkinje cells, the neurons, the white matter and the chorioid plexus of the brain. Aldolase containing mostly B-monomers were found in the proximal tubules. Aldolase isozymes particularly rich in C-monomers were seen in the nervus opticus, the pia mater, the vessels of cerebrum and the molecular layer of the cortex cerebelli.

Die LDH-isoenzyme als ursache für unspezifische tetrazoliumsalz-anfärbungen in gelzymogrammen (“nothing dehydrogenase”)

Abstract It was demonstrated that after tetrazolium salt staining of gel enzymograms of extracts containing NAD-dependent dehydrogenases, a pattern of lactate dehydrogenase ( l -lactate: NAD-oxidoreductase, E.G. 1.1.1.27) isoenzymes also became visible, although the incubation mixture contained no lactate, but only the specific substrate for the dehydrogenase examined. From the experiments reported it appears that the supporting media of the gel electrophoresis (starch, polyacrylamide, cellulose acetate) contain groups similar to lactate insolubly bound to the polymerous gel system. This is probably the case also with agar gel. This fault unavoidably occurs with all methods used. By differential staining of horizontal layers of a gel with different substrates it was shown that extracts of human tissue contained only a single anodic band of glycerol-3-phosphate dehydrogenase ( l -glycerol-3-phosphate: NAD-oxidoreductase, E.G. 1.1.1.8) and a single anodic band of glutamate dehydrogenase ( l -glutamate: NAD-oxidoreductase, deaminating, E.G. 1.4.1.3). The remaining bands that could be visualized by usual staining methods have been identified as lactate dehydrogenase isoenzymes. Confirmation was obtained by comparison with results of ion exchange Chromatography. These results offer a simple explanation for the unspecific staining of lactate dehydrogenase isoenzymes, and make it possible to avoid erroneous conclusions in the examination of multiple forms of enzymes, as seen in numerous reports recently.

Histological Examination of the Aldolase Monomer Composition of Cells from Human Kidney and Hypernephroid Carcinoma

Aldolase was specifically fixed in tissue sections by the use of antibody prepared either against aldolase A or against aldolase B. The localization of the antigen was demonstrated with the immuno-histochemical method. Aldolase A was found to be the predominant constituent in the cytoplasm of the distal tubules, the large vessels and the glomerula of normal kidney, and aldolase B in the proximal tubules. The collecting tubules and the capillaries contained a mixture of the two types. In the hypernephroid carcinoma cells only aldolase A could be found, but the capillaries within the tumor tissue did contain some aldolase B. Confirmation of these results was obtained by analysis of homogenate prepared with carefully selected tissue parts.

Ribonuclease A digestion by proteinase K.

Abstract The digestion of ribonuclease A by proteinase K yielded one major degradation product only, which could not be distinguished from ribonuclease S by electrophoretical and immunological methods. This component (ribonuclease K) possessing full catalytic activity was characterized to be (1-20/211-124) ribonuclease A . Combined action of proteinase K and trypsin on ribonuclease A leads to a significant increase of the inactivation rate which may be useful in the isolation of mRNA from polysomes.

Immunological specificity of the isoenzymes I and III of human hexokinase (ATP:D-hexose 6-phosphotransferase EC 2.7.1.1) Estimation of isoenzyme pattern by quantitative immunotechniques☆

Abstract The immunological relationship of hexokinase isoenzymes I and III from human tissues was tested with antisera made against purified forms of these enzymes. There was no evidence for crossreactivity as tested by immunoinhibition and precipitin experiments. In mixtures of the two hexokinases made from stock solutions the relative concentration of each form could be determined by specific precipitation with the homologous antiserum. When the method is applied to extracts from human tissues high concentrations of hexokinase form III were found in liver (more than 50% of total hexokinase activity), spleen (up to 50%) and lung (below 20%). From the immunological data it is concluded that the low K m hexokinases from mammalian tissues constitute a family of distinct proteins with similar catalytic properties. The general applicability of immunoinhibition methods for the evaluation of the tissue concentrations of the hexokinase isoenzymes is discussed.

On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3-phosphate dehydrogenase as tested by specific modification

A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast. As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used. The residue which is modified by the procedure described has been identified as Trp-193. There are either one or two molecules of the modifying agent being added to this tryptophan side chain. The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed. However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification. It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme. Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.

Characterosation of a highly hydrophobically modified lactate dehydrogenase

1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.