The role of the polymorphic genes apolipoprotein E and methylene- tetrahydrofolate reductase in the development of dementia of the Alzheimer type.

The gene for apolipoprotein E (APOE) is polymorphic, and its variant APOE4 is a major risk factor for the development of Alzheimer-type dementia (AD). Another risk factor for AD appears to be negative cobalamin balance, which is very common in elderly people. Cobalamin and folate are interdependent and essential components of the one-carbon metabolism. Another important component is methylenetetrahydrofolate reductase (MTHFR), the gene for which is also polymorphic. Thermolabile MTHFR (tMTHFR), a gene variant that reduces the activity of its enzyme, is common in the general population. In the present study, 75% of 140 AD patients had at least one APOE4 allele. The numbers of APOE4 and tMTHFR alleles correlated significantly with the serum folate levels, however, in opposite directions. The significance of this was augmented by an inverse correlation between APOE4 and tMTHFR. Thus, not only MTHFR but also APOE appears to be related to the one-carbon metabolism, suggesting that APOE4 and insufficient one-carbon metabolism may be synergistic risk factors for AD.

Homozygous thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis.

The gene for methylenetetrahydrofolate reductase (MTHFR) has shown polymorphism in the general human population. In its homozygous form, a C677T mutation occurs in more than 5% of the grown-up population and produces a thermolabile variant which reduces the overall enzyme activity to less than 30% of normal. We investigated patients with schizophrenia-like psychosis. If hyperhomocysteinemic, their DNA-genotype for thermolabile C677T mutation was determined. Seven of 11 patients, six males and one female, were homozygous for thermolabile MTHFR. One male patient was heterozygous and all three normal homozygotes were females. In the patients who were homozygous for the C677T mutation, the homocysteine concentrations did not respond to vitamin B12 but were normalized by folate supplementation. In the normal homozygotes, however, the homocysteine concentrations were reduced by vitamin B12 alone. Our results suggest that homozygosity for thermolabile MTHFR is a risk factor for schizophrenia-like psychosis. Possibly, this risk may be reduced by folate supplementation.

Study of the nature of recognition in molecularly imprinted polymers.

In the present study molecularly imprinted polymers (MIPs) were prepared against a series of structurally related compounds containing various numbers of pyridyl groups. The goal, to increase understanding of the mechanisms of recognition in MIPs, was achieved by comparing the patterns of retention of the imprinted compounds on the different MIPs when related to a blank (non-imprinted) polymer in a high performance liquid chromatography system. Furthermore, frontal analysis was carried out on three polymers: a blank, a pyridine-imprinted and a 4,4'-bipyridyl-imprinted polymer, to evaluate the number (Bt), average specificity and strength (dissociation constant; Kdiss) of the recognition sites. The Kdiss values of pyridine on the different polymers were in the range 0.10-0.12 M, and the amount of imprinted binding sites (Bt) 0.10-0.12 mmol/g. Kdiss values of 4,4'-bipyridyl were approximately 0.06 M, with Bt values equal to the above, except for in the anti-4,4'-bipyridyl polymer where the Kdiss was determined to be 0.02 M and Bt 0.07 mmol/g. From the results it can be concluded that multiple additive weak interactions dominate the recognition of the template molecules in these imprinted polymers.

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.

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.

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.