DNA Phosphorus Research Articles

A polynucleotide CD probe: interactions with oligomers of a polycationic amine that exhibit positive extrinsic bands at greater than 300 nm.

AbstractA set of large positive extrinsic CD bands ([θ]333 = 2.6 X 104 deg‐cm2/decimole phosphate) in the > 300 nm region as well as diminution of the intrinsic signals (θ275) have been observed in the CD spectra of various nucleic acids complexed with the achiral compound, N‐poly{α‐[N‐(4‐pyridylethylene‐4‐pyridyl‐N′‐)α′‐p‐xylyl]dibromide}‐4‐pyridylethylene‐4‐pyridinium bromide, (polymer X).1,2,5 The signal changes are attributed to the binding of polymer X chromophores isogeometrically to the DNA helix in an ordered chiral arrangement. Fractionation of polymer X gives 10 well‐separated oligomers. The oligomers were characterized by nmr. Their interactions with DNA have been investigated with respect to r(r = ratio of equivalents of polymer X charge/g‐atoms DNA phosphorus) and n (oligomer chain length). In all cases where n ≥ 1, [θ]333 increases linearly with increasing r between 0 and 0.32, and is accompanied by a corresponding decrease in [θ]275, which becomes negative as r approaches .32. Extrinsic band intensities reveal a dependence on n up to n = 5, above which increases in nonspecific binding result in a reduction in normalized band intensities. Polymer X shows a strong preference for B‐form nucleic acids and induces maximum extrinsic CD signal intensities with A‐T homopolymers. Alterations in helix hydration are believed to accompany complex formation. Inversions in [θ]275 of the octamer X‐poly(dA‐dT) complex have been attributed to the “alternating B” conformation of poly(dA‐dT).3 Similar inversions are not observed in other nucleic acid‐octamer X complexes. Visible and CD spectrometry data from competition studies in the presence of the antibiotics actinomycin D (AMD), daunomycin (DM), and distamycin A (DST) are consistent with “nonclassical” intercalation as the mode of binding, and these data place the potential binding site in or near the hydrophobic region of the minor groove. Reductions in [θ]333 with increasing urea further implicate the involvement of hydrophobic interactions in the formation of an asymmetric complex. Stabilization of the helix results in all cases as evidenced by alterations in Tm; corresponding changes, however, in cooperativity are not clearly discernable. Viscosity and light‐scattering data indicate no changes in molecular weight due to aggregation, and as such are not consistent with a transition to the ψ‐DNA upon complex formation.

Binding of monoclonal anti-native DNA autoantibodies to DNA of varying size and conformation

A microchemical assay for phosphorus was applied to the measurement of DNA in immune complexes formed with monoclonal or serum anti-DNA autoantibodies and DNA of varying size and conformation. Two monoclonal antibodies were produced by hybridomas derived from spleen cells of autoimmune MRL-lpr/lpr mice and were purified from culture fluid by affinity chromatography on columns of goat anti-mouse Ig-Sepharose. Double-helical DNA fragments were prepared by brief digestion of calf thymus DNA with micrococcal and S1 nucleases and fractionation on Sepharose 4B; their double-stranded structure was confirmed by measurement of thermal denaturation. Immune complexes were formed with monoclonal or serum antibodies and native DNA or DNA fragments or denatured DNA; the complexes were precipitated with goat anti-mouse IgG and washed, and DNA phosphorus content of the precipitates was measured. With one monoclonal autoantibody (H241), there were discontinuous increases in the amount of DNA that could be bound (and decreases in the antigen concn required for half-maximal binding) as the DNA size increased. There were especially marked increases in binding efficiency as fragment size increased from an average of 100 (range 85–105) to an average of 150 (range 105–170) base pairs, and again between 450 (range 360–620) and 600 (range 425–825) base pairs. A second monoclonal antibody (H143) did not show significant variation in binding with DNA fragments larger than 300 base pairs. With smaller fragments, the amount of DNA bound by H143 was reduced, but the DNA concn required for half-maximal binding was not. Affinities of these monoclonal antibodies were within the spectrum of human systemic lupus erythematosus serum IgG anti-DNA autoantibodies. The dependence of binding on mol. wt is important in the evaluation of these monoclonal antibodies as biochemical reagents and as potential participants in formation of immune complexes in vivo.

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei.

A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-p was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D = 6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 micrograms/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.

Comparison of the binding of some carcinogenic and non-carcinogenic cyclopenta[a]phenanthrenes to DNA in vitro and in vivo

After microsomal activation in vitro, both the strong carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one and its inactive 12-methyl isomer bind covalently to added DNA, in the ratio approximately 6:1 (1595 and 254 mu mol/mol of DNA phosphorus, respectively). Over a thousand times less binding was observed when DNA was isolated from the skin of groups of mice that had received a topical dose of 1000 nmol of these two compounds, and of the unsubstituted ketone (non-carcinogen) and its 11,12-dimethyl derivative (carcinogen), 48 h previously; the binding ratios were 458, 155, 19 and 974 nmol/mol DNA phosphorus, respectively. Covalent binding of the 11-methyl compound to mouse skin DNA, measured 48 h after topical application, was linear with dose over the range 50 - 1000 nmol. It has previously been demonstrated for groups of mice initiated with 200 or 400 nmol of this carcinogen and promoted by repeated application of croton oil that skin tumour incidences were 50 and 70%, respectively. For a topical dose of 1000 nmol of the inactive 12-methyl isomer DNA binding in skin falls in this range, equivalent to DNA binding given by approximately 340 nmol of the carcinogen. Thus although the inactive unsubstituted parent compound essentially fails to bind to skin DNA after topical application, there does not seem to be a consistent relationship between extent of DNA binding in vivo and carcinogenicity among these cyclopenta[a]phenanthrenes. However, loss of the 12-methyl adducts from skin followed a logarithmic course over the first 10 days following application, with a half life of 3.5 days. In contrast, it was previously shown that removal of the 11-methyl adducts could not be measured above the normal rate of DNA turnover for this mouse tissue (half life, 6-7 days). It is suggested that active DNA repair of the 12-methyl lesions may contribute to the lack of activity of this isomer.

Representative applications of heparin-sepharose in the removal of polyamines from biological materials.

Many experimental systems would greatly benefit from the availability of a simple and effective technique to remove polyamines from biological materials. We have examined the possibility of utilizing heparin-sepharose in the removal of polyamines from rat heart mitochondria, DNA-spermine complex, and fetal calf serum. Heparin-sepharose removes 90% of spermine adsorbed to the cytoplasmic surface of rat heart mitochondria. Heparin-sepharose almost totally removes spermine from DNA-spermine complex, leaving less than 0.003 mol spermine/mol DNA phosphorus. Heparin-sepharose is highly effective in removing spermine and spermidine (99.5 and 95% adsorbed, respectively) from fetal calf serum. Under the same experimental conditions only 50% of putrescine is adsorbed. A higher amount of resin corresponding to an increased capacity for putrescine must be used to achieve a satisfactory removal of putrescine.

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study.

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.

Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. I. Electron microscopy of ash structures.

Tipula iridescent virus (TIV) is a relatively large particle containing about 15% DNA. As shown elsewhere by X-ray microanalysis (Thomas RS, Corlett M: J Histochem Cytochem 29:394, 1981), low-temperature oxygen plasma microincineration of the virus produces a stable phosphorus oxide ash representing this DNA nearly quantitatively. Transmission electron microscopy (TEM) of entire particles after plasma incineration shows the ash confined to the viral cores, confirming the previously known general location of the nucleic acid. Examination of ultrathin-sectioned virus crystals after plasma etching or ashing shows, on a still finer scale, that the DNA is probably confined to a shell structure within the core. A fine trace of ash from the capsid, seen in some preparations, may represent a phospholipid internal membrane known to be present. The possibilities of ash pattern artifacts are discussed. Heating experiments show that the ash patterns (and native virus particles) evaporate completely at high temperature, consistent with their presumed polyphosphoric acid composition. A heat-stable ash could be formed, however, when the viral DNA became accidentally stained with iron from the steel TEM grids used--a noteworthy artifact. The present work suggests some future possibilities of the plasma microincineration technique. In particular, the ability to see directly the fine distribution of mineral concentrations in ash patterns with the full resolution of TEM should be a powerful adjunct to increase effectively the sensitivity and resolution of X-ray microanalysis of mineral constituents in biological specimens.

Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. II. Electron probe X-ray microanalysis of ash constituents.

Microincinerated, 1-micrometer sections of Tipula iridescent virus (TIV) crystals were analyzed quantitatively with a wave-length-dispersive electron microprobe to determine the chemical composition of high-resolution ash patterns previously obtained from ultrathin sections of the same specimen (Thomas RS: J Histochem Cytochem 29:379, 1981). Parallel analyses were performed on intact sections. In some cases, the same section and probe tracks were analyzed both before and after ashing. The principal elemental constituent of the ash was phosphorus, representing nearly all of the phosphorus found in the unashed sections. This confirmed the likely DNA origin of most of the ash. Other elements--sulfur, calcium, sodium, and perhaps carbon and nitrogen--found in relatively small concentrations in the ash, were partly due to either to incomplete ashing or to a particulate contaminant. Similar plasma ashing and analyses of sectioned model preparations of polymethacrylate containing dissolved triphenyl phosphate confirmed that phosphorus, by itself, could be retained as a stable ash. Analyses of polymethacrylate containing aluminum as well as phosphorus disclosed an unexpected artifact--aluminum inhibited the plasma ashing. These results suggested that the wavelength-dispersive probe, able to analyze for carbon and nitrogen as well as mineral elements, should be a generally useful tool in analyzing plasma microincineration phenomena, where macroscopic results do not apply. Relatively high beam intensity used throughout the probe analyses caused obvious damage to intact sections, particularly when they were mounted on thin-film supports. In contrast, the ash appeared quite stable. This suggested that plasma ashing of biological sections, converting them into mineralogical specimens, may be generally useful in probe analyses of mineral constituents, permitting greater sensitivity via higher beam currents, higher mineral concentrations, and lower backgrounds.

EcoRI-generated reiterated components of the rat genome I. Sequence of two (92 and 93 bp) related DNA fragments

The sequence of the 92 and 93 bp long, highly repetitive DNA fragments, isolated from EcoRI digested rat liver DNA, were determined. These fragments, designated 92 and 93, are found in equal abundance, 6.5 × 10 5 copies per haploid genome. J92 and J93 can be distinguished by their differential sensitivity to cleavage by HaeIII and HindIII, respectively, which cut the fragments at 75 and 57 bp from their mutually homologous 5'-ends. J92 and J93 are 38% and 35.4% G + C, respectively, and contain a disproportionate number of triplets complementary to stop codons in all reading frames. Three methylated sites were found in J92 while none could be detected in J93. The sequences around the m 5C sites were 5'-Py-Py-m 5C-G-Pu-Pu, except for one case where the second Py was replaced by an A. This site appeared to be hemimethylated. When J92 and J93 are placed in register from their mutually homologous 5'-ends, homology is 73% for the first 30 bp region and 63.5% for the total molecule. Thermal melting studies indicate sequence heterogeneity within J92 and J93 from substantial internal base mismatches. The sequences derived are therefore composite averages for the whole molecules. The Cot / 1 2 for the sequence was measured spectrophotometrically to be 2 × 10 −2 M/s on a DNA phosphorus basis and 2.15 × 10 −4 M/s on a mole fragment basis.

Isolation of deoxyribonucleic acid from Mycobacterium avium by rapid nitrogen decompression.

Deoxyribonucleic acid (DNA) of high molecular weight could be isolated from cells of Mycobacterium avium if the cells were exposed to nitrogen gas at 1,500 lb/in2 for 30 min and then brought to atmospheric pressure by rapid decompression. DNA isolated from the cells had a molecular weight of 4.8 x 10(6) to 17.4 x 10(6). DNA was also released into the fluid in which the cells were suspended during nitrogen decompression. One-half of this DNA, representing 3% of the total DNA phosphorus in the cells had a uniform molecular weight of 4.2 x 10(6). This DNA was linear in conformation, and removal of associated carbohydrates did not change its sedimentation rate. The biological function or significance of the 4-megadalton DNA was not determined.

Deoxyribonucleoproteins of herring sperm nuclei. I. Chemical composition.

The chemical composition of deoxyribonucleoproteins from herring sperm nuclei was analyzed and the results are summarized as follows: 1. Chemical analysis of nuclear proteins and nucleic acids revealed that arginine/P molar ratio in herring sperm nuclei is unity but the ratio of arginine residues in protamine to phosphorus in the total DNA is 0.86. 2. The deoxyribonucleoproteins were isolated and their composition showed that about 14% of the total DNA in herring sperm nuclei is free from protamine and is bound with nonprotamine proteins in the weight ratio of nonprotamine proteins to DNA of 0.25-0.30. The remaining 86% of the total DNA is combined mainly with protamine and a small amount of nonprotamine proteins; the weight ratios of protamine and nonprotamine proteins to DNA are 0.75 and 0.08, respectively. In the latter complex, the molar ratio of arginine residues in protamine to phosphorus in DNA is unity.

Modification du métabolisme du phosphore dans les feuilles et les racines de froment (Triticum aestivum) sous l’action des herbicides triaziniques

Summary Atrazine and promethrine were applied at 1 ppm during 20 h and at 0.02 ppm during 48 h to the roots of twelve day old wheat seedlings in nutrient solution. RNA, DNA and acid-soluble mineral phosphorus were determined in the roots and in the leaves 1, 2, 3, 5 and 7 days after the treatment. In the leaves of seedlings treated with lethale dose, acid-soluble mineral phosphorus increased progressively and reached 124–127% of the control at the seventh day. RNA decreased by 9–16% to 23–24% while DNA remains practically unchanged. In the roots, acid-soluble phosphorus decreased by ± 10%, RNA by 23–25 to 31–34% and DNA by 18–24 to 35–46%. The application of sublethale dose does not involve increasing of nucleic acid biosynthesis neither in the leaves nor in the roots.

X-ray photoelectron spectroscopy of DNA · Pt complexes Evidence of O 6(Gua) · N 7(Gua) chelation of DNA with cis-dichlorodiamine platinum(II)

The binding energies of nitrogen, oxygen, phosphorus, chlorine and Pt in several DNA · Pt (ll) complexes are reported and discussed. The nitrogen band of DNA is slightly shifted upon complexation with Pt. Oxygen binding energies in the complexes studied clearly show that cis-Pt(NH 3) 2Cl 2 forms a specific chelate N 7(Gua) · O 6(Gua) with DNA as opposed to trans-Pt(NH 3) 2 Cl 2 and the other Pt compounds which react only with the N 7(Gua) site of DNA.

The induction of azaguanine-resistant mutants in cultured Chinese hamster cells by reactive derivatives of carcinogenic hydrocarbons

A study was made of the induction by two chemically reactive hydrocarbon derivatives of mutations to 8-azaguanine (8-AzG) resistance in the Chinese hamster cell line V79-379A. The compounds, 7-bromomethylbenz[ a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[ a]anthracene (7-BrMe12MeBA), were chosen since they had exhibited a considerable variation in carcinogenicity in animal experiments 10. The use of radioactively-labelled compounds enabled the extents of reaction of the derivatives with cellular macromolecules to be determined. Following treatment with the bromo-derivatives, the induced mutation frequency increased arithmetically with the number of cell divisions which occurred prior to the addition of azaguanine, and reached a maximum after 3–4 divisions. With still longer expression times, the mutant yield declined sharply. The time required to achieve 3–4 cell divisions was dependent on the dose of mutagen used. To allow comparison between mutant yields after different mutagen doses, azaguanine was added to the dishes after the cells had undergone 3 divisions. A plot of induced mutation frequency against extent of DNA reaction for both agents indicated that no significant mutation resulted from that extent of reaction corresponding to the “shoulder” on the survival curves. At somewhat higher levels of reaction, both compounds induced about 5.0·10 −5 mutants per μmole hydrocarbon derivative reacted per mole DNA phosphorus. At still higher reaction levels, an abrupt increase in mutant yield per extent of reaction with DNA occurred, 7-BrMeBA and 7-BrMe12MeBA inducing 1.3·10 −3 and 3.0·10 −4 mutants per μmole per mole DNA phosphorus reaction, respectively. The results are discussed in relation to those of others with X-ray, UV and other chemical agents, and a model is proposed which considers the role of DNA repair processes in mutation induction. The reported difference in the carcinogenicity of the two hydrocarbon derivatives studied was not paralleled by their mutagenicity in the present system.

The induction of azaguanine-resistant mutants in cultured chinese hamster cells by reactive derivatives of carcinogenic hydrocarbons

A study was made of the induction by two chemically reactive hydrocarbon derivatives of mutations to 8-azaguanine (8-AzG) resistance in the Chinese hamster cell line V79-379A. The compounds, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe12MeBA), were chosen since they had exhibited a considerable variation in carcinogenicity in animal experiments10. The use of radioactively-labelled compounds enabled the extents of reaction of the derivatives with cellular macromolecules to be determined. Following treatment with the bromo-derivatives, the induced mutation frequency increased arithmetically with the number of cell divisions which occurred prior to the addition of azaguanine, and reached a maximum after 3–4 divisions. With still longer expression times, the mutant yield declined sharply. The time required to achieve 3–4 cell divisions was dependent on the dose of mutagen used. To allow comparison between mutant yields after different mutagen doses, azaguanine was added to the dishes after the cells had undergone 3 divisions. A plot of induced mutation frequency against extent of DNA reaction for both agents indicated that no significant mutation resulted from that extent of reaction corresponding to the “shoulder” on the survival curves. At somewhat higher levels of reaction, both compounds induced about 5.0·10−5 mutants per μmole hydrocarbon derivative reacted per mole DNA phosphorus. At still higher reaction levels, an abrupt increase in mutant yield per extent of reaction with DNA occurred, 7-BrMeBA and 7-BrMe12MeBA inducing 1.3·10−3 and 3.0·10−4 mutants per μmole per mole DNA phosphorus reaction, respectively. The results are discussed in relation to those of others with X-ray, UV and other chemical agents, and a model is proposed which considers the role of DNA repair processes in mutation induction. The reported difference in the carcinogenicity of the two hydrocarbon derivatives studied was not paralleled by their mutagenicity in the present system.

Activity of alkaline phosphatase and carbonic anhydrase in male and female zinc-deficient rats.

Severe zinc deficiency was induced in male and female rats by feeding them a purified diet containing 0.5 mg zinc per kg of diet. Symptoms of zinc deficiency included severe growth retardation, loss of hair and scaly paws. Zinc-metallo enzymes were measured in kidney, stomach, and intestine of male rats and kidney and intestine of female rats. Alkaline phosphatase per mg DNA-phosphorus (DNAP) and per gram wet weight of tissue was lowered compared to ad libitum fed controls in all organs of both male and female animals. Carbonic anhydrase was lowered in all organs of male rats. It was only slightly lowered in kidney of female rats and was higher than in controls in stomach of female rats. DNA-phosphorus per gram wet weight was higher than in the controls in all organs of male animals and in the kidney of female animals. Stomach of female animals showed the same DNAP-concentration in experimental and control animals.

Changes in nitrogen and nucleic acid contents of the liver and muscle of preruminant lambs given high and low protein diets

Twenty-one crossbred lambs (aged 2-5 days) were allotted at random to three groups. A control group of six lambs was slaughtered immediately. Six lambs were given a low protein diet (diet A) and nine lambs a high protein diet (diet B). Subgroups of three lambs given diet A were slaughtered after 2 and 6 weeks on experiment; subgroups of three lambs given diet B were slaughtered after 2, 4, and 6 weeks. At slaughter the deoxyribonucleic acid (DNA), ribonucleic acid, and nitrogen (N) contents of the liver and vastus lateralis muscle were determined. It was possible, from a knowledge of the DNA phosphorus contents of these organs at different ages, to determine the relative increase or decrease in cell numbers per organ and in cell size. A comparison of (1) the comparative slaughter method and (2) the N/DNA phosphorus ratio method, for estimating the protein gain or loss from the liver and muscle, showed poor agreement between the two methods. The reason for the discrepancy was the change in the total DNA phosphorus content of the liver and muscle during growth.

Circular dichroism of aminoacridines bound to DNA.

AbstractThe binding curves of 1‐, 2‐, 3‐, and 9‐aminoacridine and proflavine on native DNA and the circular dichroism (CD) spectra of the bound cations have been determined under the same conditions. The variation of the CD spectra with the amount (r) of aminoacridine bound per DNA phosphorus was of two main kinds: (1) the rotational strength of those aminoacridines which possess a 3‐amino group depended markedly on r and decreased to relatively small values (or zero) at zero r; or, (2) the rotational strength changed relatively little with r and tended to a finite value at zero r. The relevance of these observations is discussed with respect to interelation models of the complexes and with respect to possible explanations of the basis of this induction of optical activity.

A chemical procedure for the replacement of some of the bases in DNA by marker molecules

Salmon sperm DNA was alkylated in aqueous solution at pH 7.0–7.2 using N, N-diethyl-2-chloroethylamine. Subsequent treatment of the alkylated DNA with 3H-or 14C-labelled methoxyamine at pH 7.2 led to incorporation of the labelled methoxyamine to an extent of one methoxyamine molecule per 15–16 atoms of DNA phosphorus. The total recovery of DNA phosphorus throughout the entire procedure was 85%. Nonalkylated DNA treated in a similar manner showed small incorporation of radioactivity.

Enzymic changes in rabbit and rat mammary gland during the lactation cycle.

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.