Membrane selectivity versus sensor response in hydrogenated amorphous silicon CHEMFETs using a semi-empirical model.

Toxic amides, such as acrylamide, are potentially harmful to Human health, so there is great interest in the fabrication of compact and economical devices to measure their concentration in food products and effluents. The CHEmically Modified Field Effect Transistor (CHEMFET) based on amorphous silicon technology is a candidate for this type of application due to its low fabrication cost. In this article we have used a semi-empirical model of the device to predict its performance in a solution of interfering ions. The actual semiconductor unit of the sensor was fabricated by the PECVD technique in the top gate configuration. The CHEMFET simulation was performed based on the experimental current-voltage curves of the semiconductor unit and on an empirical model of the polymeric membrane. Results presented here are useful for selection and design of CHEMFET membranes and provide an idea of the limitations of the amorphous CHEMFET device. In addition to the economical advantage, the small size of this prototype means it is appropriate for in situ operation and integration in a sensor array.

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method.

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase.

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy.

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.

The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis.

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((2)H(2)O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients epsilon of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1)cm(-1) at 1625, 1605, and 1365 cm(-1), respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V(max), K(m), and K(cat)) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4mM, and 806.36 s(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.

Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy.

A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.

A monoclonal antibody specific for Pseudomonas aeruginosa amidase.

Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography. The MAb was specific for amidase from P. aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme.

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability.

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Purification and characterization of monoclonal antibodies against the free alpha-subunit of human chorionic gonadotrophin.

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.

Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme.

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

Production and characterization of a specific rubisco monoclonal antibody, and its use in rubisco quantification during Zantedeschia aethiopica spathe development.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified from leaves of Zantedeschia aethiopica and used to immunize female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A random selected IgG2a subclass MAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL-4B, with a recovery of 84.3% and it was apparently homogeneous on native PAGE. The monoclonality of the purified MAb was determined by IEF. The MAb was highly specific for Rubisco from leaves of Z. aethiopica as determined by Western blotting and was used to determine the concentration of Rubisco protein by enzyme-linked immunoadsorbent assay (ELISA), at three distinct stages of Z. aethiopica spathe development and in the leaf. The results suggest de novo synthesis of Rubisco during the spathe regreening, which could explain, at least in part, the increase of photosynthetic activity observed during regreening.

Glucose 1- and 2-oxidases from fungal strains: isolation and production of monoclonal antibodies.

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.

Monoclonal antibodies against urease from Canavalia ensiformis.

Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canavalia ensiformis were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. All culture wells exhibited hybrid growth and 25% of these (ie 45 culture wells) contained anti-urease activity. Two positive hybrid cells were cloned twice by the limiting dilution method and three hybridoma clones (B6F, C4F and B18) secreting monoclonal antibodies were selected at random for purification and characterisation purposes. All three cell lines secreted monoclonal antibodies of IgM class which were purified by gel filtration chromatography on Sephacryl S-200 column with a final recovery of 85% and a purification factor of about 18. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 920,000 Da. mAbs were highly specific for jack bean urease as determined by Western blotting. The affinity constants (K) for these mAbs ranged from 10(8) to 10(9) l mol-1. mAb B6F inhibited about 65% of urease activity whereas C4F and B18 stimulated the enzyme activity slightly by 20%. The presence of 2-mercaptoethanol in incubation mixtures protected urease from inactivation by B6F. Urease inactivation by B6F could be reversed by addition of 2-mercaptoethanol which reactivated most of the partially inactive enzyme. Gel filtration chromatography of purified urease exhibited two protein peaks with M(r) values of 290,000 and 90,000 Da which revealed antibody activity. This result suggests that the mAb B6F recognizes the trimeric as well as the monomeric forms of urease.

Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa.

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.

One-step purification and properties of catalase from leaves of Zantedeschia aethiopica.

Catalase (E.C 1.11.1.6) was purified from leaves of Zandedeschia aethiopica to apparent homogeneity by a one-step hydrophobic interaction chromatography on a phenyl Sepharose CL-4B column. The purified enzyme preparation was obtained with a final recovery of enzyme activity of about 61% and a specific activity of 146 U/mg protein. The purified enzyme ran as a single protein band when analyzed both by native PAGE and SDS-PAGE corresponding to an Mr of 220,000 Da, which consists of 4 subunits with identical Mr of 54,000 Da. The pI of purified enzyme was found to be 5.2 by isoelectric focusing on ultrathin polyacrylamide gels. The purified catalase has an optimum temperature of activity at 40 degrees C, whereas it is stable between 0 degrees and 50 degrees C. As regards pH, the enzyme has an optimum activity at pH 7.0 and it is stable in the range pH 6-8. The absorption spectrum of the purified enzyme exhibited 2 peaks at 280 nm and 405 nm.

One-step affinity purification of urease from jack beans.

Jack bean (Canivalia ensiformis) urease (EC3.5.1.5) was purified in one-step by ligand affinity chromatography using epoxy-activated Sepharose 6B-urea. The yield of the purified enzyme was about 80% with a specific activity of about 500 U/mg of protein. The enzyme was apparently homogeneous when analyzed by SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the stained band of urease activity. The affinity column could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either acetamide or semicarbazide as affinity ligands were also found to be useful for the isolation of urease.

Purification and properties of peroxidase from Pinus pinaster needles.

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.

Improved purification and properties of glucose dehydrogenase from Bacillus subtilis.

Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography. The specific activity of the purified preparation was about 460 units/mg of protein with a final recovery of enzyme activity of about 75%. The affinity column could be regenerated and reused again several times. The purified enzyme appeared to be homogeneous when analyzed both on SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the activity stain. ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers. In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers.

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions.

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.