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A sensitive and convenient biosensor for detection of heavy metal salts has been developed. The method is based on the effects of heavy metal salts on the catalytic activity of L-lactate dehydrogenase (LDH) in solution or coimmobilized with L-lactate oxidase (LOD) on an oxygen electrode. At metal concentrations below 100 microM, the kinetic behavior, with the LDH substrate NADH, showed a competitive inhibition with high affinity during the first 10 s. With increased incubation time, irreversible first order inactivation with respect to enzyme concentration was observed. This irreversible inactivation of LDH in solution was dose dependent. The efficiencies obtained for the different heavy metal salts were: HgGl2 > AgNO3 > Pb(COOCH3)2 > CuSO4 > ZnCl2. HgCl2 and AgNO3 were effective in the nanomolar range while the other metal salts acted at the micromolar level. LDH is protected by saturating amounts of substrate NADH against the effects of the heavy metal salts studied. The pKs for LDH catalytic activity and inactivation by heavy metal salts were similar. The results suggest binding of the heavy metal salts to the enzyme active site. Except for lead acetate, all heavy metal detection was in the range of European norms. For AgNO3, CuSO4 and HgCl2, the sensor limit of detection reached the European norm values whereas with ZnCl2 it was well below. The immobilization of LDH considerably decreased the amount of enzyme consumed by permitting repetitive assays. The efficiency of inactivation by the heavy metal salts was reduced in comparison with LDH in solution. Restoration of activity of the inactivated immobilized enzyme was obtained with DTT, EDTA, KCN and NADH treatment. This opens up possibilities for detection of toxic compounds using simple procedures suitable for assays in a variety of monitoring conditions in environmental and food pollution control.

In a culture medium E. coli produces L -lactate as a metabolite. L -lactate enzyme sensors were assembled and applied to a crude E. coli growth medium for L -lactate determination. The amount of L -lactate was used as an index of the cell number. In the standard solution, L -lactate oxidase (LOD) sensor gave a linear response from 5 to 300 μM L -lactate. Determination of L -lactate in bacteria cultures of different absorbance (A) was possible from an absorbance at 600 nm, A600=0.01 and higher. L -lactate measurements were not modified when the LOD electrode was associated with L -lactate dehydrogenase (LDH) in solution. In contrast, the bi-enzyme sensor consisting of LOD coimmobilized with LDH on the electrode, lowered the detection limit to 30 nM L -lactate by amplification of measurements via coupled reactions and substrate recycling. Responses were obtained in growth medium from A=0.0005 (5×105 bacteria ml−1). In comparison, the standard spectrophotometer cell estimation was not reliable below A=0.02. The L -lactate detection limit can be decreased further by incubating bacteria with glucose for 1 to 2 h. Measurements of L -lactate by oxygen consumption could be performed in the presence of bacteria and growth medium components. The recycling enzymatic electrode kept 80% of its initial activity with storage overnight in a buffer, at 4°C, during 2 weeks. Such sensors permit fast and sensitive procedures for L -lactate and E. coli assays. The procedure does not disturb the developing cultures as relatively small sample volumes from 1 to 500 μl and no cells are needed for L -lactate determination.

Inactivation by paraoxon of free and immobilized acetylcholinesterase, in the presence of various amounts of different organic solvents, and with choline oxidase immobilized on an oxygen electrode was studied. With acetylcholinesterase in solution it was found that 5% cyclohexane enhances paraoxon detection when compared to the detection without solvent. In these conditions, as low as 10 −9 m paraoxon can be detected. Free acetylcholinesterase was more sensitive to paraoxon than immobilized acetylcholinesterase, depending on the nature and concentration of solvent (500-fold in the presence of 5% cyclohexane). The stability of the biosensor in continuous use and stored at room temperature was at least 3–4 weeks with triton X100 and bovine serum albumin addition. The sensitivity of paraoxon detection with free acetylcholinesterase in the presence of 5% cyclohexane corresponds to 0·2 ppb paraoxon, 10-fold below the legal limit admitted in the European Economic Community. The same approach could be applied for detection of low amounts of different organophosphorus or carbamate pesticides.

Covalent immobilization of L-lactate oxidase (LOD) with L-lactate dehydrogenase (LDH) on a film tightly bound to an oxygen electrode, for rapid and sensitive L-lactate measurements, is described. Regeneration of L-lactate by substrate recycling provided an amplification of the sensor response, making it possible to decrease the detection limit of L-lactate from 10 μM to 20 nM. The apparent K m for L-lactate with the LOD-LDH coupled reaction was 1 μM, compared with 3 mM when utilizing only LOD. Linearity was obtained from 20 to 300 nM with both enzymes, whereas with LOD alone it was from 10 μM to 1 mM. Optimization of the biosensor was obtained with an increase in LOD and LDH film loading and low L-lactate concentration. The enzymes covalently bound to the film stabilized the biosensor (half life 8 weeks) for over 400 measurements. Low L-lactate excreted by E. coli bacteria metabolism can be assayed in turbid culture medium without pretreatment by the amplified L-lactate detection.

Twenty new 2-(cyanoalkyl)tetrazoles (15 and 16) and twenty new 2-(hydroxyalkyl)tetrazoles (17 and 18) were synthesized and investigated in vitro for their abilities to inhibit selectively rat brain monoamine oxidase (MAO) B over MAO A. Most of them were MAO B inhibitors and those bearing a substituted 4-(arylmethoxy)phenyl group in the position 5 of the tetrazole ring had IC50 values between 8 microM for 18d and 2 nM for 16a (30 nM for lazabemide) with a selectivity toward MAO B of 37,000 for 16a. The reversibility of their inhibitory activity was demonstrated by in vitro dialysis tests. The 5-[4-(phenylmethoxy)phenyl]-2-(2-cyanoethyl)tetrazole (16a) its derivative 16h and the 5-[4-(phenylmethoxy)phenyl]-2-(2-hydroxyethyl)tetrazole (18a) and its derivative 18h were found to be potent, in vitro selective, and competitive MAO B inhibitors. Tetrazole 16a can be considered one of the most active and selective competitive MAO B inhibitors known up to now. This compound was selected for ex vivo experiments and was shown to be a strong and reversible MAO B inhibitor with a short duration of action after oral administration at 5 mg/kg. The structure-activity approach gives rise to the great importance of lipophilicity over electronic effects of the compounds in these series.

Immobilized oxidases were bound on the surface of an affinity membrane and mounted on an oxygen electrode. These biosensors were used for heavy metal salt measurements. After immobilization on the enzymes, first order kinetics of inactivation were observed. Surface immobilization increases the sensitivity by a factor of 10, compared to reticulation of the enzyme in a gelatin matrix. After immobilization, 50% inactivation was observed with 20 μ m HgCl 2 for L-glycerophosphate oxidase and 50 n m for pyruvate oxidase. Restoration of activity after HgCl 2 treatment is feasible, but neither complete nor reproducible. To reuse the biosensor, L-lactate dehydrogenase (LDH) from rabbit muscle in solution was coupled to immobilized L-lactate oxidase (insensitive to heavy metal salts). LDH (particularly inexpensive) was replaced after each measurement. The I 50 in phosphate buffer was 1 μ m for HgCl 2 and 0·1 μ m for AgNO 3 ; with other heavy metal salts, no inhibition was observed below 500 μ m . In Tris buffer, the I 50 was 10 μ m for CdCl 2 and ZnCl 2 , 50 μ m for Pb-acetate and 250 μ m for CuSO 4 . The use of different enzymes and buffers may allow measurement of specific heavy metal salts.

Eighteen new 5-aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues were prepared and evaluated in vitro for their inhibitory properties on monoamine oxidase (MAO) types A and B. The most active compounds in these series acted preferentially against MAO B with IC50 values in the range of 1.8-0.056 μM. The 5-(4-biphenylyl)-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one 23 and its oxadiazolethione analogue 33 were found to act as potent, selective and competitive MAO B inhibitors with a slight slow-binding character. Both compounds inhibited MAO A in a classical competitive manner. According to their Ki (MAO B) values of 2.6 and 4 × 10−8 M, respectively, and their Ki (MAO A)/Ki (MAO B) ratios of 270 and 500, respectively, 23 and 33 can be placed among the most active and selective competitive MAO B inhibitors known up to now. The structure-activity relationships are discussed.

Twenty new 2-(cyanoalkyl)tetrazoles (15 and 16) and twenty new 2-(hydroxyalkyl)tetrazoles (17 and 18) were synthesized and investigated in vitro for their abilities to inhibit selectively rat brain monoamine oxidase (MAO) B over MAO A. Most of them were MAO B inhibitors and those bearing a substituted 4-(arylmethoxy)phenyl group in the position 5 of the tetrazole ring had IC50 values between 8 microM for 18d and 2 nM for 16a (30 nM for lazabemide) with a selectivity toward MAO B of 37,000 for 16a. The reversibility of their inhibitory activity was demonstrated by in vitro dialysis tests. The 5-[4-(phenylmethoxy)phenyl]-2-(2-cyanoethyl)tetrazole (16a) its derivative 16h and the 5-[4-(phenylmethoxy)phenyl]-2-(2-hydroxyethyl)tetrazole (18a) and its derivative 18h were found to be potent, in vitro selective, and competitive MAO B inhibitors. Tetrazole 16a can be considered one of the most active and selective competitive MAO B inhibitors known up to now. This compound was selected for ex vivo experiments and was shown to be a strong and reversible MAO B inhibitor with a short duration of action after oral administration at 5 mg/kg. The structure-activity approach gives rise to the great importance of lipophilicity over electronic effects of the compounds in these series.

Eighteen new 5-aryl-1,3,4-oxadiazol-2(3H)-one derivatives and sulfur analogues were prepared and evaluated in vitro for their inhibitory properties on monoamine oxidase (MAO) types A and B. The most active compounds in these series acted preferentially against MAO B with IC50 values in the range of 1.8-0.056 μM. The 5-(4-biphenylyl)-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one 23 and its oxadiazolethione analogue 33 were found to act as potent, selective and competitive MAO B inhibitors with a slight slow-binding character. Both compounds inhibited MAO A in a classical competitive manner. According to their Ki (MAO B) values of 2.6 and 4 × 10−8 M, respectively, and their Ki (MAO A)/Ki (MAO B) ratios of 270 and 500, respectively, 23 and 33 can be placed among the most active and selective competitive MAO B inhibitors known up to now. The structure-activity relationships are discussed.

Coimmobilization of l -lactate oxidase (LOD) and l -lactate dehydrogenase (LDH) on a film bound to an oxygen electrode makes it possible, by recycling l -lactate and pyruvate, to lower their detection limit to 10 nM. With LOD immobilized alone and LDH in solution, easy LDH measurements were performed. The detection limit was 1 IUl−1. Similarly, this mixed biosensor, using an LDH sensitive to mercurials, was used for p-chloromercuribenzoate (PCMB) detection. Coimmobilization of LOD + LDH with an NAD+-dependent dehydrogenase allows the measurements of all dehydrogenases. NAD+ is recycled. The detection limit was 50 nM. Applications can be found in medicine, the food industry, and the environment.

Thirty-three new 5-[4-(benzyloxy)phenyl]-1,3,4-oxadiazol-2(3H)-one derivatives including related analogues, designed as inhibitors of monoamine oxidase type B (MAO B), were synthesized and investigated both in vitro and ex vivo for their abilities to inhibit selectively rat brain MAO B over MAO A. Three inhibitors were found to act as reversible, highly potent, and selective MAO B inhibitors, namely the nitrile derivative 5-[4-(benzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (12a) and two closely related homologues, the corresponding oxadiazolethione 13a and the alcohol 14b. Their IC50 (MAO B) values are in the low nanomolar range of 1.4-4.6 nM and their selectivities, estimated by the ratio of IC50 values (A/B), are from 3200 to > 71,400. Compound 12a exhibited the highest activity against MAO B. Its IC50 was evaluated to be 1.4 nM with a quasitotal selectivity (> 71,400) toward this enzyme. In ex vivo studies, 12a showed a reversible and short duration of action. MAO B was markedly inhibited with the oral dose of 1 mg/kg without any alteration of MAO A, and the inhibition almost did not exceed 24 h. Its ED50 (1 h after oral administration) was evaluated to be 0.56 mg (1.7 mumol)/kg. Remarkably, MAO A was not affected at doses as high as 1500 mg/kg, po. In addition, no apparent toxicity or behavioral anomaly was observed during the treatment even at the maximum administrated dose. SAR studies emphasize the existence of three binding sites to the enzyme with a special importance of the terminal phenyl. Analysis of the inhibition kinetics indicated that 12a acts in a two-step mechanism as a competitive, slow, and tight-binding inhibitor of MAO B with a Ki value of 0.22 microM and an overall Ki* value at equilibrium of 0.7 nM.

Publisher Summary This chapter focuses on biosensors for heavy metal salt measurements with immobilized enzymes on a Clark electrode. Most immobilized enzymes are sensitive to 1 mM heavy metal salts. After immobilization at the surface of an affinity membrane (Ultrabind from Gelman), some enzymes are much more sensitive to lower concentrations of mercury salts. In a study described in the chapter, 50% inactivation (I50) was obtained with HgCl2 after 2 min incubation. Inactivation of the enzymes was measured with a Clark electrode whose sensitive tip was covered with the immobilized enzyme film. Addition of substrate induces oxygen consumption, which is inhibited by the heavy metal salt. For pyruvate oxidase, the sensitivity was further increased reaching 10 nM when the incubation with HgCl2 was performed in the absence of thiaminepyrophosphate-Mg. The biosensors are very easy to use, and the assay lasts less than 10 s. Applications can be found for solving pollution problems of water.

Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATPdriven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern. The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites. Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes. The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.

Monoamine oxidase (MAO) assay specificity based on substrate specificity was investigated by substrate competition experiments. 10 μM serotonin (5-HT) and 5 μM β-phenylethylamine (PEA) were found to ensure total substrate specificity for, respectively, MAO types A and B. Twenty-five 2-aryl-4 H -1,3,4-oxadiazin-5(6 H )-one derivatives were synthesized and tested in vitro for their inhibitory effects on MAO A and B. Most of them inhibited preferentially MAO B. The 2-(4-biphenylyl)-4-(2-cyanoethyl)-4 H -1,3,4-oxadiazin-5(6 H )-one 32 was the most efficient MAO B inhibitor and acted as a competitive inhibitor on the two enzymes. Its K i values for MAO A and B were 11 and 0.15 μM, respectively. Structure-activity relationships suggest that these oxadiazinones should interact with a hydrophobic site and a nucleophilic site on MAO B for binding, while the functional group of the N-4 substituent should compete with the substrate for the active site of the enzyme.

Contrary to the generl view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro-organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less that 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non-limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate. NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.

Monoamine oxidase (MAO) assay specificity based on substrate specificity was investigated by substrate competition experiments. 10 μM serotonin (5-HT) and 5 μM β-phenylethylamine (PEA) were found to ensure total substrate specificity for, respectively, MAO types A and B. Twenty-five 2-aryl-4 H -1,3,4-oxadiazin-5(6 H )-one derivatives were synthesized and tested in vitro for their inhibitory effects on MAO A and B. Most of them inhibited preferentially MAO B. The 2-(4-biphenylyl)-4-(2-cyanoethyl)-4 H -1,3,4-oxadiazin-5(6 H )-one 32 was the most efficient MAO B inhibitor and acted as a competitive inhibitor on the two enzymes. Its K i values for MAO A and B were 11 and 0.15 μM, respectively. Structure-activity relationships suggest that these oxadiazinones should interact with a hydrophobic site and a nucleophilic site on MAO B for binding, while the functional group of the N-4 substituent should compete with the substrate for the active site of the enzyme.

The melibiose permease system of E. coli K12 has been explored using a strain deficient in lactose permease: 300 P. The accumulation of 1-S-methyl-beta-D-thiogalactopyranoside (TMG) was observed. The uptake system was inducible by melibiose and a number of analogs at 30 degrees C. At higher temperatures the differential rate of synthesis decreases until becoming negligible at 42 degrees C. The uptake tends toward a steady state which corresponds to an accumulation several hundredfold over the sugar concentration in the medium. At a given temperature the steady state level was proportional to the initial rate of uptake whatever the degree of induction and the substrate concentration. Lowering the temperature decreased the initial rate of uptake but increased the steady state level. This uptake system was pH dependent with better efficiency at pH 8. It was also dependent on the presence of sodium or lithium ions active at 5 mM whereas potassium at 170 mM enable only about half maximal uptake. The uptake in a medium with choline chloride was less than one fifth of optimal activity. Addition of Li+ brought about half maximal activation at approximately 0.5 mM. The activation consists mainly in a decrease of apparent Km. The emphasis of this study was put on the similarities and differences with lactose permease which is able to transport the same sugar to approximately the same extent. Inducer specificities and substrate specificities were compared and a method of measuring the two activities in the same cells was devised.

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD(+)-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD(+) required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD(+) from NADH. This NAD(+) can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10-30% of the initial activity.Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.

1. Exposure of submitochondrial particles to phospholipases impaired the rate of oxidation as well as phosphorylation. Phospholipase A and phospholipase C were more inhibitory to phosphorylation and to NADH oxidation than to succinate oxidation. 2. At low concentrations of phospholipase A the inhibition of phosphorylation was prevented to a considerable extent by the presence of large amounts of bovine serum albumin preferably during both digestion and assay. At larger concentrations of phospholipase A only slight protection was observed by addition of serum albumin. 3. Exposure of submitochondrial particles to phospholipase C from Clostridium welchii in the presence of Mg++ or Mn++ inhibited the 32Pi-ATP exchange and the P:O ratio by about 30 to 60%. Addition of coupling factors restored phosphorylation associated with oxidation in these particles. Additional exposure of the digested particles to phospholipase C from Bacillus cereus in the presence of Zn++ further depressed the 32Pi-ATP exchange which was no longer restored by coupling factors. Addition of phospholipids partially restored the 32Pi-ATP exchange in these extensively digested particles. Similar effects were noted after prolonged exposure to phospholipase C from B. cereus alone, provided both Mn++ and Zn++ were present. 4. Oxidative phosphorylation was also severely impaired after exposure of submitochondrial particles to phospholipase C from B. cereus in the presence of Mn++ and Zn++. A partial restoration of the P:O ratio was achieved by reconstitution with phospholipids.

Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p -nitrophenyl- σ -galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p -nitrophenyl- α -galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn 2+ , a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved.

1. Exposure of bovine heart mitochondria to purified preparations of phospholipase A or phospholipase C resulted in losses of phosphorylation capacity, whereas respiration was not impaired. 2. The inhibitory effects of low concentrations of phospholipase A on phosphorylation were prevented or reversed by bovine serum albumin. At higher concentrations of phospholipase A neither bovine serum albumin nor lecithin were effective in restoring phosphorylation. 3. Exposure of mitochondria to phospholipase C resulted in a loss of Ca++ translocation, a partial depression of oxidative phosphorylation, and a partial loss of organically bound phosphorus. 4. Phospholipase C-treated mitochondria were resistant to the uncoupling action of Ca++ or K+ plus valinomycin. Addition of nigericin together with valinomycin and K+ was required to abolish phosphorylation associated with oxidation in phospholipase C-treated mitochondria. These responses resemble those of submitochondrial particles rather than those of mitochondria. In electron micrographs, phospholipase C-treated mitochondria looked like submitochondrial particles packaged in mitochondria. 5. A partial restoration of Ca++ translocation after phospholipase C digestion was achieved by addition of either egg lecithin or of large amounts of bovine serum albumin.