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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.

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.

Raw, unprocessed coconut supports the growth of salmonellae as well as that of other enteric bacteria, salmonellae being particularly resistant to subsequent desiccation. Original contamination is not due to carriers or to polluted water supplies, but to contact with bacteria-containing soils followed by dispersion via infected coconut milk and shells. Pasteurization of raw coconut meat in a water bath at 80 C for 8 to 10 min effectively killed such bacteria, did not injure the product, and provided a prophylactic method now widely used by the coconut industry.

An immobilized three-enzyme system, malate dehydrogenase (EC 1.1.1.37)-citrate synthase (EC 4.1.3.7)-lactate dehydrogenase (EC 1.1.1.27), was investigated as a model for the rate of oxalacetate production and utilization in mitochondria. Lactate dehydrogenase is included to mimic the NADH-utilizing system of mitochondria. This three-enzyme system was immobilized in three different ways (1) on Sephadex G-50 (surface coupling), (2) on Sepharose 4B (internal-external coupling), and (3) entrapped in polycrylamide gel. The rate of citrate production from malate, NAD(+), and acetyl CoA was determined continuously in a flow system. Up to about 100% rate enhancements were observed when the immobilized system was compared to identical systems of free enzyme. An even more pronounced increase of rate of up to about 400% compared to the soluble system was measured after addition of pyruvate (to reoxidize formed NADH). These results are interpreted in relation to microenvironmental changes of oxalacetate production and the possible organization of enzymes of the Krebs cycle.