Search PubMed⌕ Search

Biomedical subjects

G Pfleiderer

Publications and source records attributed to G Pfleiderer.

At least 37 records · Page 2Linked to original sources
PubMed1984

[pH-induced inactivation of glycerol dehydrogenase from Bacillus megaterium].

The kinetic of the reversible dissociation of glycerol dehydrogenase from Bacillus megaterium in slightly alkaline medium was measured by biochemical, chemical and physical methods. The dissociation is followed by changes in the secondary structure and can be prevented by addition of NAD or increased potassium chloride concentration. Crosslinking by suberimidate, but not by monofunctional imido esters, shows a high stabilization against alkali, urea or heat inactivation caused by hindrance of dissociation.

Bacillus megaterium↗
PubMed1983

Synthesis and application of a fluorescent imido ester for specific labelling of amino groups in proteins.

2-(5'-Dimethylaminonaphthalene-1'-sulfonamido)methylimidic acid methyl ester has been synthesized for fluorescence labelling of amino groups in proteins. The incorporation of the dansyl group serving as an extrinsic fluorescent probe is determined spectrophotometrically. Glucose dehydrogenase (beta-D-glucose: NAD(P+) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium having a reactive lysine residue which belongs to the active site has been labelled. To give proof of the selectivity of the modification, the enzyme preparation having 1.3 dansyl groups per subunit has been digested with trypsin and the major labelled peptide has been isolated and sequenced.

Amines↗
PubMed1983

Amino acid sequence of an insect chymotrypsin from the larvae of the hornet, Vespa orientalis.

The complete amino acid sequence of the endopeptidase II from the larvae of the hornet, Vespa orientalis, has been determined. The enzyme is a single polypeptide chain of 216 residues. The protease is a serine endopeptidase. When aligned for optimal homology to the trypsin related proteases, the insect endopeptidase displays 37% identity with bovine chymotrypsin. The structure of the hornet protease is the first reported for a serine endopeptidase from an insect.

Amino Acid Sequence↗
PubMed1983

Importance of tyrosine for structure and function of mitochondrial 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.

Animals↗
PubMed1983

Solid-phase direct immunoassay for serum intestinal and placental alkaline phosphatase.

Antibodies against placental alkaline phosphatase (PAP) share antigenic determinants with the intestinal isoenzyme (IAP) and vice versa. Both isoenzymes can be found as part of the total activity of alkaline phosphatase (AP) in the serum. Using antibody-coated polystyrene tubes, a simple and sensitive immunoassay was developed which allows the quantitative determination of IAP or PAP without interference of the cross-reacting isoenzyme. The presence and amount of IAP depends on the ABO blood group, secretory status and the oral fat intake. The serum IAP in healthy fasted individuals was found up to 8 U/I in secretors of blood group O and B and below 1 U/I in non-secretors and blood group A donors. In screening tests of various pathological sera. IAP was found elevated up to 100 U/I in idiopathic hyper-AP-aemia and some liver cirrhosis patients.

ABO Blood-Group System↗
PubMed1983

[Isolation and characterization of glycerol dehydrogenase from bacillus megaterium].

Glycerol dehydrogenase of high purity was isolated from Bacillus megaterium. The enzyme has a pH optimum of 9 and dehydrogenizes in presence of NAD+ glycerol as well as 1,2-propanediol and, to a smaller extent, erythritol. The Michaelis constant for glycerol is 1.4 X 10(-3)M and for NAD+ 3 X 10(-4)M. With the application of different methods (density gradient centrifugation, gel electrophoresis and gel chromatography) relative molecular masses of 156 000-160 000 were ascertained. In the dodecyl sulfate electrophoresis we found a molecular mass of 38 000 per subunit, so that the native enzyme is existent in the tetrameric form. By means of fluorescence titration one NADH binding site per subunit could be estimated. The amino-acid composition was determined. The enzyme is stable only in the presence of high thiol concentrations. Heavy metal ions and chelating agents inhibit the activity of the enzyme. Zinc, presumably, is the natural heavy metal in the active center, but other divalent metal ions can also activate the apoenzyme. With the help of chemical modification reactions, the probable presence of one essential thiol group and one essential tyrosine residue per subunit could be demonstrated.

Amino Acids↗
PubMed1982

Development and evaluation of a new solid-phase direct immunoenzyme assay for prostatic acid phosphatase.

In this assay we used polystyrene-tube-attached rabbit antibodies against prostatic acid phosphatase (PAP) that had been purified to homogeneity from human prostate. The amount of immunoreactive acid phosphatase was determined directly by its enzymic activity in the solid-phase-bound immune complex. The detection limit was 0.05 U/L (0.13 microgram/L), the CVs between 4.3 and 10.8%. Investigating the organ specificity of PAP, we found that some cross-reacting acid phosphatase activity could be so measured in human kidney, leukocytes, and platelets, all of which probably contribute to the circulating "prostatic" acid phosphatase that normally is present in serum. Diurnal and day-to-day variations in serum PAP activity were as much as 100% in healthy subjects. Individuals without prostatic diseases (n = 92) had values for serum PAP activity up to 0.36 U/L (0.94 microgram/L), in an age-independent distribution; patients with benign prostatic hyperplasia (n = 62) showed values up to 0.48 U/L (1.25 micrograms/L). With PAP activity of 0.38 U/L or 1.0 microgram/L (90th percentile of the prostatic group) as the upper limit of "normality," overall sensitivity (stages A-D) for detection of prostatic cancer in 33 essentially untreated patients was 65%. Examples for the followup of therapy of prostatic cancer by measurement of serum PAP with this assay are described.

Acid Phosphatase↗
PubMed1980

Change in alkaline phosphatase isoenzyme pattern in urine as possible marker for renal disease.

The human kidney contains two types of alkaline phosphatase (AP) isoenzymes: a hepatic type of AP and an intestinal-like AP. Intestinal-like AP, measured by immunotitration techniques, is a minor component (1 to 4%) of the total AP activity. It is found only in the particle-free fraction (cytoplasm) and is located, with immunofluorescent techniques, in some of the proximal convoluted tubules. Urinary AP activity is found after high-speed centrifugation in the supernatant (x 100,000g), as well as in the sediment, and may be extracted from the sediment after solubilization with n-butanol. Both types of these renal isoenzymes contribute to urinary AP activity. Biochemical characterization (effect of inhibitors, thermostability, denaturing with urea, and so on) revealed that urinary intestinal-like AP and renal intestinal-like AP are identical. Both, however, have been distinguished as multiple forms of AP from the small intestine. Most of the urinary AP activity of healthy persons (22 volunteers) was found in the sediment and consisted of liver-type AP. Urinary AP of patients with diseases, after application of potentially nephrotoxic drugs or during rejection episodes of renal allografts, contains little sediment activity, but it contains increased amounts of urinary intestinal-like AP.

Alkaline Phosphatase↗
PubMed1980

Factors affecting the activity of immobilized enzymes, I. Diffusional limitation.

The measured (apparent) specific activities of immobilized lactate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, alkaline phosphatase and chymotrypsin decrease with increasing activity loading and increase with decreasing particle diameter and with increasing substrate concentration. The observed inactivation is therefore concluded to be due to diffusional limitation. Real specific activities are not greatly affected by the immobilization, as has been demonstrated after enzymic digestion of the matrix. In the case of lactate dehydrogenase and of malate dehydrogenase real specific activities are not altered by a variation of the number of bonds to the carrier.

Alkaline Phosphatase↗
PubMed1979

Immunotitration of alkaline phosphatase isozymes in normal and pathological urine.

Isoenzyme patterns of alkaline phosphatase are determined quantitatively in extracts of human kidney as well as in human urine by means of immunotitration technique. Both media contain two types of AP isoenzymes: liver and intestinal like AP. Intestinal AP is located as a minor component of total AP activity (1-4%) in particle-free fraction of the kidney. Urinary AP activity is found after high speed centrifugation in supernatant (100,000 Xg) as well as in the 100,000 Xg sediment and can only be made soluble from the latter by n-butanol treatment. Intestinal AP in urine is concentrated in the supernatant while in sediment the isoenzyme pattern resembles to that of kidney. Urine of normal persons contains most of AP activity in the sediment and consists mainly of liver type AP. Urinary AP of patients with renal diseases or after application of cytotoxins contains little sedimentable activity, mainly intestinal AP.

Alkaline Phosphatase↗
PubMed1978

The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.

Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.

Alkaline Phosphatase↗
PubMed1978

Enzymatic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro.

An endopeptidase from the larvae of the hornet Vespa crabro has been purified to homogeneity. The enzyme has been characterized with respect to molecular weight, amino acid compositon, and amino- and carboxyl-terminal sequences. The catalytic properties of the hornet protease are similar to those of bovine chymotrypsin with respect to inactivation by phenylmethanesulfonyl fluoride and carbobenzoxyphenylalanine chloro ketone and preferential peptide bond cleavage at aromatic amino acid residues. In contrast to bovine chymotrypsin, the hornet protease is not inhibited by the basic pancreatic Kunitz inhibitor, soybean inhibitor, or chicken ovomucoid. The molecular weight, as determined by several independent methods, was found to be 14 500. The protease is a single-chain protein containing two disulfide bonds. The terminal sequences are: NH2-Ile-Val-Gly-Gly-Ile-Asp.....Gly-Lys-Tyr-Pro-Tyr-Gln-Val-Ser-Leu-Arg-COOH.

Amino Acid Sequence↗
PubMed1978

Human diploid lung fibroblast cell lines WI 26 and WI 38 exhibit isozyme shift of alkaline phosphatase after viral transformation.

The testing of the type of membrane associated alkaline phosphatase (EC 3.1.3.1) by immunotitration has revealed that there is a shift from a liver-like type of alkaline phosphatase in normal cell cultures of the human diploid fibroblast cell strains WI 26 and WI 38 to a placenta-like variant in cultures of the same cell strains after the transformation by the DNA-tumor virus SV 40, the WI 26 SV 40 and the WI 38 SV 40 cell lines. The immunologically detectable switch-over has been confirmed by measuring the apparent Michaelis constant and the heat stability of the AP from normal and transformed cells. Liver-like AP is heat labile and has an apparent Michaelis constant for p-nitrophenylphosphate (about 4.0 X 10(-4) M). The placenta-like AP shows heat stability and a lower apparent KM (about 2.2 X 10(-4) M). The appearance of the so-called Regan enzyme of AP in some human tumors in vivo is discussed in this connection.

Alkaline Phosphatase↗
PubMed1978

[Lactate dehydrogenase. An example of the development of modern enzymology].

Lactate dehydrogenase is the prototype of an oligomeric, intracellular enzyme. Its primary, secondary, tertiary, and quaternary structures are now well known. This fact, especially with regard to the occurrence of isoenzymes, has effected a great progress in the understanding of the structure-function relationship of enzymes. The historical development of modern enzymology is described, together with discussions of the various methods and problems of enzymology.

Animals↗
PubMed1978

Synthesis and application of a new bifunctional amidination reagent.

Methyl 4-hydroxy-3-nitrobenzimidate hydrochloride (I) is presented as a newly developed amidination reagent. It is synthesized in two steps from commercially available 4-hydroxybenzonitrile. Its incorporation into proteins can be easily determined spectroscopically. The activities of the three enzymes lactate dehydrogenase (pig heart), alkaline phosphatase (calf intestine) and tryptophan synthase (E. coli) were not greatly reduced after modification with the reagent. The nitro groups can be reduced by mild treatment with Na2S2O4 without alteration of the enzymatic activity. The newly formed aromatic amino groups can further be made to react at lower pH than the native amino groups.

Alkaline Phosphatase↗