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Biomedical subjects

K Mosbach

Publications and source records attributed to K Mosbach.

At least 145 records · Page 8Linked to original sources
PubMed1978

Covalent binding of an NAD analogue to liver alcohol dehydrogenase resulting in an enzyme-coenzyme complex not requiring exogenous coenzyme for activity.

1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.

Adenosine Monophosphate↗
PubMed1977

Enzyme thermistor determination of glucose in serum using immobilized glucose oxidase.

An enzyme thermistor assay for serum glucose is described. The glucose present in the sample is reacted in a small column containing glucose oxidase immobilized to controlled pore glass (single thermistor device). The heat produced in the primary reaction is measured directly in the column without any need for coupling reactions. The useful linear range is 0.01-0.45 mM glucose, permitting 50-fold dilution of serum samples. Advantages are low enzyme cost, due to the immobilization, insensitivity for the color or any turbidity of the sample, and no requirement for coenzyme or any ancillary reaction. Improved sensitivity and extended linear range (0.01--0.9 mM) can be attained through a secondary reaction using catalase. The application to glucose analysis of a split-flow enzyme thermistor equipped with a reference column to eliminate unspecific heat effects is also described. The enzyme thermistor determinations were also compared with a spectrophotometric continuous flow technique using a small column with immobilized glucose oxidase and 4-aminoantipyrine and phenol as color reagents.

Blood Glucose↗
PubMed1977

Aspects of microenvironmental compartmentation. An evaluation of the influence of restricted diffusion, exclusion effects, and enzyme proximity on the overall efficiency of the sequential two-enzyme system malate dehydrogenase--citrate synthase in its soluble and immobilized form.

Soluble bifunctional enzyme aggregates have been prepared by cross-linking the sequential enzymes malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) using glutaraldehyde. The kinetic behaviour of this two-enzyme system in its aggregated and non-aggregated form was studied both in free solution and immobilized on Sepharose beads. This study was undertaken in order to distinguish between the following two factors which may account for the increased efficiency found in general in co-immobilized consecutive two-enzyme systems: (a) closer proximity between the participating enzymes and (b) establishment of a favourable microenvironment (such as higher local intermediate concentration caused by increased diffusional hindrance in the gel phase). It was found that in spite of a reduction of the distance between the two enzymes in the aggregated form by an estimated factor of 10(3), no kinetic advantage (shorter lag phase or higher steady-state rate) could be detected compared to the corresponding system with the two enzymes not linked to each other. However, both systems immobilized to Sepharose reached the steady-state rate of citrate formation almost immediately, in contrast to the corresponding free systems which exhibited pronounced lag phase. These results indicate that, at least in the above systems and under the conditions given, diffusional hindrance in the gel phase of the intermediate oxaloacetate, which is present in rate-limiting concentrations, is the dominant cause of the observed higher efficiency in immobilized systems.

Citrate (si)-Synthase↗
PubMed1977

Thermometric enzyme linked immunosorbent assay: TELISA.

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Catalase↗
PubMed1977

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.

The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.

Alcohol Oxidoreductases↗
PubMed1977

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B.

Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin.

Animals↗
PubMed1977

Application of cyanide-metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase.

The application of the enzyme thermistor in the analysis of cyanide in standard solutions as well as in blast furnace waste water is described. The heat signal is generated in the conversion of cyanide, catalyzed by the immobilized enzymes rhodanese (E.C. 2.8.1.1) and injectase (E.C. 4.4.19). Using the combination of cyanide-metabolizing enzymes and the enzyme thermistor unit, assays down to 20 microM cyanide can be carried out. Linear relationships were obtained at 20-600 microM cyanide for injectase and 20-1000 microM for rhodanese. The stability at 27 degrees C of the heat response was initially decreased, but soon stabilized at about 80% of the initial value and remained so for at least 200 hr. The technique was easily adapted to continuous analysis, applicable to environmental control (e.g., a "cyanide guard") with a response time at present within 2-3 min after a sudden change in cyanide concentration has appeared.

Carbon-Sulfur Lyases↗
PubMed1976

Effects of subunit interactions on the activity of lactate dehydrogenase studied in immobilized enzyme systems.

Rabbit muscle lactate dehydrogenase (LDH) was coupled to Sepharose in such a way that each molecule is expected to be attached via only one subunit. Dissociation of the bound active enzyme by several methods all yielded immobilized subunit derivatives which were inactive. These derivatives were capable of regenerating activity by interacting specifically with subunits in solution formed transiently during renaturation. This ability to peck up soluble subunits is lost fairly rapidly upon storage of the immobilized subunits. Similarly, LDH subunits attached to Sepharose via disulfide bonds were found to be inactive. When these subunits were detached from the matrix by mild reduction with mercaptoethanol, activity was regenerated. The kinetics of this reactivation process suggests that reassociation is required for appearance of activity. All these results can be interpreted as showing that subunit interactions are essential for LDH activity.

Animals↗