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M DeLuca

Publications and source records attributed to M DeLuca.

At least 73 records · Page 4Linked to original sources
PubMed1978

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.

Flavin Mononucleotide↗
PubMed1977

Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.

Chromatography, Affinity↗
PubMed1977

Thermally induced myocardial preservation and necrosis in deprived fetal mouse hearts.

Cultured fetal mouse hearts deprived of oxygen and glucose were used to examine the effect of temperature on the mechanical, biochemical, and ultrastructural responses of the deprived myocardium to assess the utility of this in vitro model for studying myocardial necrosis, preservation, and repair. After 4 h of deprivation at 4, 24, 37, or 42 degrees C, 1) beating had ceased;2) ATP levels were decreased by 22% for 4 degrees C insults, 69% for 24 degrees C, 89% for 37 degrees C, and 97% for 42 degrees C; 3) CPK and LDH levels were unchanged; and 4) ultrastructural changes were observed. After 24 h of recovery from deprivation, 1) beating resumed, except for 42 degrees C;2) ATP levels were 102% of control for 4 degrees C; 99% for 24 degrees C; 62% for 37 degrees C; and 4% for 42 degrees C; 3) LDH content was decreased by 0% at 4 degrees C; 6% at 24 degrees C; 35% at 7 degrees C; and 70% at 42 degrees C; and 4) CPK content decreased similarly. Hypothermia protected deprived myocytes while hyperthermia accelerated cell necrosis. Combining deprivation with thermal insult in this in vitro model provides a spectrum of myocardial damage for studying the effect of interventions on repair processes and on metabolic changes in jeopardized myocardium.

Adenosine Triphosphate↗
PubMed1977

Detection of perioperative myocardial damage after coronary artery bypass graft surgery.

In order to evaluate methods for detecting peri-operative myocardial damage we studied 41 patients before and serially following coronary artery bypass graft surgery utilizing the 12-lead ECG, serum MB-CPK measurements, and 99mTc pyrophosphate myocardial scans. Six of the 41 patients (15%) developed persistent new Q waves after surgery. Six other patients demonstrated ischemic ST-T wave changes that persisted for 48 hours or more. Mean total MB-CPK released was highest for the group with new Q waves [1598+/-545 (SE) I.U./L-hr] as compared to the group with ischemic ST-T wave changes 708+/-65 I.U./L-hr) or the group with no ECG changes (262+/-47 I.U./L-hr). Ten patients (24%) has positive postoperative pyrophosphate scans consistent with myocardial infarction. The three techniques were compared in these 41 patients utilizing 465 I.U./L.-hr as the upper limit of normal MB-CPK released after uncomplicated coronary bypass surgery (no ECG changes, negative scan). Five patients with ischemic ECG changes had a positive scan and high MB-CPK; six patients with no ECG changes had high MB-CPK but a negative scan; and one patient with high MB-CPK and new Q wave had a negative scan. We conclude 1) new Q waves on ECG underestimate the incidence of myocardial damage after coronary artery surgery; 2) MB-CPK alone overestimates the incidence of infarction; and 3) a combination of the three techniques is the best means for detecting myocardial damage after coronary artery bypass graft surgery.

Adult↗
PubMed1976

Immobilization of bacterial luciferase and FMN reductase on glass rods.

Bacterial luciferase and NAD(P)H: FMN oxidoreductase isolated from Beneckea harveyi were covalently linked via diazotization to arylamine porous glass beads which had been cemented onto plain glass rods. These immobilized enzymes are individually active and also function to produce light via a coupled reaction utilizing NADH or NADPH. These enzymes have properties similar to the soluble forms with regard to pH and substrate optima and also exhibit linearity in peak intensity of the initial flash of light emitted as a function of NADH or NADPH concentration. Linearity with NADH is obtained in the range of 1 pmol to 50 nmol, and between 10 pmol to 200 nmol for NADPH. The bound enzymes are stable and reusable. This immobilized system offers a rapid and inexpensive m

Enzymes, Immobilized↗
PubMed1975

Fetal mouse hearts: a model for studying ischemia.

A new experimental model for the study of two important aspects of ischemia, namely, oxygen and substrate deprivation, is proposed: the intact, beating fetal mouse heart in organ culture. This model offers long-term stability, ease and reproducibility of preparation, and the ability to manipulate experimental conditions. Hearts deprived of oxygen and glucose ceased beating immediately. After 3-4 hr of deprivation, biochemical and ultrastructural changes consistent with ischemic injury were evident. These include depletion of ATP and glycogen levels, loss of cytoplasmic enzymes, and extensive swelling and disruption of mitochondrial structure. Glucose and insulin partially protected against ATP depletion. Upon resupply of oxygen and glucose , beating resumed immediately, ATP levels rapidly increased to control levels and, consistent with this, mitochondrial structure returned toward normal. During the recovery phase autophagic vacuoles containing damaged mitochondria and myofibrils were seen, indicating that repair mechanisms were activated. Consistent with this, the proportion of lysosomal enzymes that were present in the nonsedimentable fraction of the tissue homogenate increased. We conclude that the cultured fetal mouse heart is a model useful for studying myocardial responses to anoxia and/or substrate deprivation and for assessing interventions designed to limit damage or to stimulate repair after ischemic injury.

Acetylglucosaminidase↗
PubMed1974

Kinetic properties of the isoenzymes of human creatine phosphokinase.

Studies of the three human creatine phosphokinase (EC 2.7.3.2) isoenzymes, MM, MB, and BB, show that important differences exist in substrate dependency of the reaction rates. A method was developed to study these properties in which the ATP formed in the reverse reaction was measured by means of firefly luciferase. With substrate conditions at which the isoenzymes showed substantial differences in activity the method could be used for the detection of changes in the isoenzyme pattern of the serum of patients with an acute myocardial infarction. The MB isoenzyme appearing in this condition could be detected quantitatively.

Adenosine Triphosphate↗
PubMed1971

Mechanism of oxidative carbon dioxide production during Renilla reniformis bioluminescence.

The oxidation of luciferin catalyzed by sea pansy luciferase results in the emission of light. Molecular oxygen is required and carbon dioxide is produced. When the reaction occurs in the presence of H(2) (18)O, both of the oxygens of the carbon dioxide are labeled. One of the oxygens arises from the nonenzymic exchange of the ketone group of the substrate; the other oxygen is incorporated during the enzymic oxidation of the luciferin. When the reaction is carried out in the presence of (18)O(2), neither of the oxygens of the carbon dioxide is labeled. Thus the source of oxygen in the carbon dioxide is water. A mechanism for the oxidative reaction is proposed.

Carbon Dioxide↗