Joachim Müller

An experimental verification of the theory of diffusion limitation of immobilized enzymes

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was covalently bound to Sepharose beads. Kinetic measurements with the substrate p-nitrophenyl phosphate gave the following results. Effectiveness factors were decreasing with increasing bound activity, decreasing substrate concentration, and increasing particle radius of the beads. With decreasing effectiveness factors, the apparent Michaelis constants were decreasing. Preparations with high bound activities did not obey Michaelis-Menten kinetics at substrate concentrations much higher than the apparent Michaelis constants. The experimental results accorded quantitatively with the theory of diffusion limitation (Engasser, J.M. (1978) Biochim. Biophys. Acta 526, 301-310).

Analysis of mast cells in rheumatoid arthritis and osteoarthritis by an avidin-peroxidase staining

SummaryAs demonstrated by labeling with peroxidase, avidin was found to bind selectively and distinctly to mast cell granules. Inhibition studies suggested that avidin is bound by heparin. Based on this new mast cell staining procedure, mast cell distribution in the inflamed synovium of rheumatoid arthritis (RA) and osteoarthritis (OA) has been investigated. In the subsynovial layer, a significant decrease in mast cell numbers was observed in RA-synovium when compared with OA-synovium. This decrease correlated with the presence of lining cell ulcers and granulation tissue and can be interpreted as the result of mast cell degranuation induced by complement-mediated or immune complex-triggered mechanisms.

Stability of dehydrogenases III. malate dehydrogenases

Cytoplasmic and mitochondrial malate dehydrogenases from pig and chicken were studied by chemical modification of amino groups, hybridization of immobilization. Determination of thermal stability was used to characterize the different species. Modification of amino groups was found to decrease thermal stability especially when neutralization of the positive charges occurred. Decreased thermal stability correlated with decreased reassociation of immobilized monomers and modification in the monomeric state completely inhibited reassociation. Thus some lysines seem to be implicated within the subunit contacts. Active monomers of the mitochondrial forms as demonstrated earlier (Jürgensen, S.R., Wood, D.C., Mahler, J.C. and Harrison, J.H. (1981) J. Biol Chem. 256, 2383-2388) were found to display unaltered kinetic properties. From hybridizations the mechanism of thermal denaturation of malate dehydrogenases was concluded to contain a rate-limiting cooperative transaction of both monomers within the dimer, as was found earlier for tetrameric lactate dehydrogenase (Müller, J. (1981) Biochim. Biophys. Acta 669, 210-215 and Müller, J. and Klein, C. (1981) Biochim. Biophys. Acta 671, 38-41).

Distribution of lysozyme in synovial tissue of patients with osteoarthritis and rheumatoid arthritis demonstrated by different enzyme histochemical methods.

SummaryLysozyme-producing cells were analysed by enzyme histochemistry in paraffin sections of synovial tissue of 60 patients with rheumatoid arthritis (RA) and 20 patients with osteoarthritis (OA). For lysozyme detection three enzym histochemical systems — peroxydase-antiperoxydase, alkaline phosphatase and biotin-avidin — were used in parallel experiments. Lysozyme was found to be produced by polymorphonuclear cells, mononuclear phagocytes and part of synovial lining cells. All types of lysozyme-producing cells were increased in RA compared with OA. Subgrouping of RA synovitis according to histomorphological criteria allowed the demonstration of an inverse relationship between the number of lysozyme-producing cells and the grade of proliferation of fibroblasts, called mesenchymoid transformation by Fassbender [19]. The different methods of lysozyme detection differed in specificity and sensitivity. The immunoenzymatic staining of lysozyme allows specific and quantitative evaluation of phagocyting cells in RA and OA.

In Situ Evaluation of Immunoglobulin Synthesis of Synovial Plasma Cells in Rheumatoid Arthritis and Osteoarthritis by Plug Photometry

Synovial membranes of patients with osteoarthritis--a disease with unaltered immunoregulation--and rheumatoid arthritis were immunostained for the different immunoglobulin isotypes using the PAP-method and a two-layer alkaline phosphatase technique. Randomly selected plasma cells with positive immunostaining were measured by plug photometry. The average extinction of the single plasma cell is considered to be indicative of the immunoglobulin synthesis of the measured plasma cell and the isotype under study. When we compared both disease entities, patients with rheumatoid arthritis were characterized (i) by higher average extinction values for all Ig-isotypes except IgG (ii) by a higher rate of high producer plasma cells. From this data, we conclude that immunophotometry is a new in vivo method for discriminating two types of synovitis (i) one without increased Ig-synthesis at the single cell level and (ii) a second one with increased Ig-synthesis.

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs.

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.

Stability of lactate dehydrogenases. I. Chemical modification of lysines

The lysine residues of lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) can be amidinated by methyl-4-hydroxy-3-nitrobenzimidate to introduce nitrophenolate anions. This modification results in lowered thermal stability, as does acetylation. The conversion of these groups into uncharged aminophenol groups without further modification of the enzyme itself stabilizes the enzymes from pig heart and muscle and from chicken muscle, as does acetamidination, but the unusually stable enzyme from chicken heart reverts only to the stability of the native form. The results allow for the following conclusions. Destabilization is brought about at many points at the surface of lactate dehydrogenases by neutralization of positive charges. Stabilization, in contrast, is concluded to be due to modification of one lysine at position 241 of the sequence. This lysine must have been changed to arginine during the evolution of heart-type lactate dehydrogenases in going from lower to higher reptiles. This exchange has been conserved in the enzymes from the hearts of birds and therefore the enzyme from chicken heart is very stable and cannot further be stabilized by modification of lysines. From X-ray structure analysis, the stabilization by exchange of Arg for Lys at position 241 or by amidination is explained by the formation of additional ion pairs with aspartic acid57 of the Q-related subunits.

Stability of lactate dehydrogenase: II . Hybrids and geometric isomers

Hybrids of lactate dehydrogenases from pig heart and muscle and from chicken heart and pig heart were obtained by the freeze-thaw method [1,2]. Ion-exchange chromatography of the resulting mixtures of hybrids yielded unusual elution patterns, i.e., the 2 + 2 hybrids (HP2MP2 and HC2HP2) were eluted in two separate peaks. These subforms were concluded to result from partial resolution of the three geometric isomers. The hybrids of chicken heart and pig heart lactate dehydrogenase showed three distinct levels of stability. The characteristics temperatures of denaturation were 61.5 degrees C for HP4, HCHP3 and HC2HP2I; 71 degrees C for HC2HP2II and HC3HP and 76.5 degrees for HC4. The resistance towards thermal denaturation thus seemed to be governed by the least stable dimer within the tetrameric enzyme. The arrangement of stabilities of the dimers was in excellent agreement with the number of additional ion pairs between Arg241 (chicken) and Asp57 (chicken and pig) [3] within the Q-contact areas. The rate-determining step of thermal denaturation of lactate dehydrogenase was concluded to comprise the distortion or dissociation of one or two Q-contacts of the tetramer.

Dissociation of mitochondrial malate dehydrogenase

The kinetics of the dissociation reaction under acidic conditions of the dimeric pig and chicken mitochondrial malate dehydrogenases (EC 1.1.1.37) have been studied. The dissociation of the pig enzyme is completely reversible. The pK for dissociation determined by light-scattering measurements agrees within experimental error with the pK value of 5.25 measured for a tyrosine-carboxylate pair. The rate constants for the dissociation reaction and for the protonation process of this tyrosine are in close agreement. Thus, the tyrosine-carboxylate pair can be used as indicator of the dissociation reaction. The dissociation of the chicken enzyme proceeds around pH 4.5 at a much lower rate. A true equilibrium between dimer and monomers is not found, since the monomer gradually unfolds at this pH. The monomers of both enzymes, pig and chicken mitochondrial malate dehydrogenase, show the same stability towards acid. The difference in stability of the dimeric forms, therefore, must be due to an altered subunit contact area.

Importance of tyrosine for sturcture and function of mitochondiral malate dehydrogenases

Mitochondrial malate dehydrogenase from pig and chicken both contain one tyrosine/subunit with highly red-shifted spectrum, most probably involved in a hydrogen bond with a carboxylate group. The spectral changes of this tyrosine can be used as an indicator for alkaline denaturation, acid transition and coenzyme binding. Acid transition is coupled with breaking of this bond by protonation as monitored by loss of absorbance at 290 nm. Activity is lost and fluorescence intensity is increased at slightly higher pH, thus indicating increased mobility of the indicator and most probably of the whole protein prior to protonation of the indicator-tyrosine.