Purification and biochemical properties of genetically defined malate dehydrogenase in maize

Malate dehydrogenase of maize exists in multiple molecular forms (isozymes). In strain W64A, two soluble forms (s-MDH), five mitochondrial forms (m-MDH), and two glyoxysomal forms (g-MDH) were found in etiolated seedlings. The s-MDHs and m-MDHs were prepared in highly purified form. Using these purified isozymes, experiments with reducing agents (100 mm mercaptoethanol), low pH (2.0), and high salt cocn (7.5 m guanidine-HCl), along with genetic data, have eliminated the possibility of conformational alterations as an explanation for MDH multiplicity in maize; the MDH isozymes are genetically determined. Biochemical properties for each of the seven MDH isozymes were examined. Molecular weight, pI, pH optimum, thermolability, and K_m for oxaloacetate, malate, NAD, and NADH at different pH values were determined for each isozyme. Different kinetics of substrate inhibition (oxaloacetate) and coenzyme inhibition (NAD) were observed for the different isozymes. Effects of NAD analogs, chelating agents, reducing agents, metal ions, and TCA cycle acids on the enzymatic activity of these isozymes were tested. Based on the physical and kinetic properties observed, the maize malate dehydrogenase isozymes can be classified into four groups: s-MDH¹; s-MDH²; the two most anodal m-MDHs; and the three most cathodal m-MDHs. Since strain W64A is highly inbred, our data along with our previous and simultaneous genetic analysis suggest that multiple genes are involved in the expression of maize malate dehydrogenase isozymes.     

Crystal and molecular structure of [N,N′-ethylenebis(methyl-2-amino-1-cyclopentenedithiocarboxylato)]copper(II): A quantitative relationship between tetrahedral distortion and redox potentials in copper N2S2 compounds and the relevance to type I copper(II) centers

The crystal and molecular structure of the copper(II) complex of the N₂S₂ tetradentate ligand, ethylenebis(methyl-2-amino-1-cyclopentenedithiocarboxylate), was solved at room temperature by a single crystal x-ray diffraction study. The complex crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 7.739(1) Å, b = 13.893(2) Å, c = 17.096(3) Å, V = 1838(1) ų, ?_observed = 1.56 g cm^?3 and ?_calculated = 1.57 g cm^?3 for a molecular weight of 434.2, and Z = 4. Diffraction data were collected with a Syntex P$\text{1}$ diffractometer using graphite-monochromatized Cu (λ = 1.5418 Å) radiation. The heavy atoms were located from a Patterson synthesis; all other nonhydrogen atoms were located using difference Fourier techniques, and hydrogen atoms were placed in calculated positions. Final refinement resulted in discrepancy indices of R = 0.067 and goodness of fit of 2.92 for all 995 reflections (5° < 2θ < 100°) greater than three times their standard deviation. The molecules are monomeric and well separated. Bond distances in the two ”halvesldquo; of the ligand are sufficiently different to suggest that different resonance structures exist in each portion. This agrees with the rhombic symmetry displayed by the frozen glass esr spectrum of the compound (_xx ≠ g_yy). The dihedral angle between the planes defined by the CuN₂ and CuS₂ planes is 20.0°, indicating a rather distorted inner coordination sphere. The copper(II)-copper(I) reduction potentials found for this compound and the trimethylene and tetramethylene analogs were determined to be ?1.01, ?0.79, and ?0.64 V respectively. A quantitative relationship between tetrahedral distortion and redox potentials is obtained, and these results are discussed in terms of ”blueldquo; copper(II) sites in proteins. Trends in CuS and CuN bonding patterns in the same three compounds are discussed with regard to the short CuS (cys) bond distance in plastocyani Finally, a brief discussion of the optical spectra of these three compounds, their variation, and their significance with respect to tetrahedral symmetry in copper(II) protein sites is presented.     

A spectral investigation of the copper(II) complex of the antitumor compound bleomycin

A thorough spectral investigation of the copper(II) complex of the antitumor compound, bleomycin, has been carried out in solution employing optical, difference optical, electron spin resonance, and circular dichroism techniques. The optical spectrum of a pH = 7 solution of the 1:1 complex between copper(II) and bleomycin is characterized by a broad weak band in the visible region (λ_max = 610 nm) that cannot be resolved and intense ultraviolet bands at 317 (? = 2800), 327 (shoulder), 250 (? = 4700), and 257 nm (shoulder). The circular dichroism spectrum in the visible region shows the broad and weak visible absorption band contains at least three components (558, 675, and 880 nm) that are likely to be “d-d” in origin. The electron spin resonance spectrum is characteristic of a tetragonal d⁹ copper(II) system showing no rhombic distoritions at X-band frequencies (g_x = g_y ± 0.002). The spin Hamiltonian parameters for the pH = 7.0 solution corrected for second order effects are A_∥ = 177 × 10^?4 cm^?1, A_⊥ ? 15 × 10^?4 cm^?1, g_∥ = 2.214, g_⊥ = 2.039. Most interesting was the observation of extra hyperfine splitting due to endogenous nitrogen coordination in a 30% glycerol glass (A^N = 12.0 × 10^?4 cm^?1). That pattern is best interpreted as a seven-line sequence associated with three liganded nitrogens. A dramatic change in all spectral properties occurs when the pH of the copper(II)-bleomycin complex is lowered to 2.5. All these data taken together suggest a CuN₃O coordination complex in solution. Details and justifications as well as a discussion of the limitations of the interpretations are presented.     

Inhibition of sexual maturation by a urinary pheromone in male prairie deer mice

Male prairie deer mice (Peromyscus maniculatus bairdii) were treated from weaning until 8 weeks of age with chemical stimuli from conspecifics or with control substances. Growth of the testes and seminal vesicles was retarded in males that were reared in contact with soiled bedding material transferred from cages of adult males. No additional inhibition resulted from the physical presence of an adult male either continuously or for 1 hr per day. Application of urine collected from adult males to the noses of young males retarded seminal vesicle growth. Removal of the olfactory bulbs of males at 3 weeks of age blocked the inhibitory influence of urine on sexual maturation. Exposure to urine from adult females did not alter the growth of the reproductive organs in young males. The ability of a male deer mouse to retard the sexual maturation of young male conspecifics (Bediz, G. M., and Whitsett, J. M., 1979, J. Comp. Physiol. Psychol.93, 493–500) appears to be a consequence of chemical stimuli excreted in its urine.     

Purification and partial characterization of alcohol dehydrogenase from wheat.

Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of Triticum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 × 10^?2m for ethanol and 1 × 10^?4m for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.     

Effects of morphine on visual evoked responses recorded in five brain sites.

The effects of morphine on averaged evoked responses to visual stimulation were examined in specific brain structures relevant to pain, analgesia, tolerance and motor disturbances. Permanent electrodes (60 μ in diameter) were implanted stereotaxically in the central gray, mesencephalic reticular formation, caudate nucleus, parafasicular-centromedian complex and the lateral geniculate body as a control site. Visual evoked responses were obtained in unanesthetized, unrestrained rats prior to and following the administration of morphine in successive doses of 1, 5, 10 and 30 mg/kg and 1 mg/kg of naloxone (a morphine antagonist). The parafasicular-centromedian complex and the reticular formation exibited a progressive increase in response amplitude to increased dose of morphine. These effects were reversed by naloxone. In this study the parafasicular-centromedian complex was found to be the most sensitive structure to morphine, displaying the largest changes in response amplitude as a result of morphine administration.     

Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture.

Developing chick skeletal muscle undergoes an isozymic shift from type K pyruvate kinase to type M during development. A major increase in pyruvate kinase activity follows the isozymic shift, resulting in at least 40-fold higher specific activities by adulthood. Similar isozymic changes occur in primary cultures of myogenic cells from 12-day-old chick embryos. Cultures initially contain only type K pyruvate kinase. Type M appears by the fourth day of culture and accounts for 80–90% of the activity by the eleventh day. Type M did not accumulate when cell fusion was prevented by removing Ca²⁺ from the growth medium or when protein synthesis was inhibited by cycloheximide.     

Interaction between low-affinity cupric ion and human methemoglobin

Human hemoglobin has been shown to contain a high- as well as a low-affinity binding site for cupric ion on each of its constitutent beta chains. The copper that is bound to the low-affinity site has been implicated in the selective oxidation of the beta hemes. In the present work a low-affinity binding site for cupric ion has been located within 10 A of the heme iron in human hemoglobin. It is suggested that the proximal histidine is involved in the binding of copper at this site and that it participates in the oxidation of heme iron and reduction of cupric ion.     

Tubulin synthesis during ciliogenesis in the mouse oviduct.

We reported earlier that tubulin levels increase in the developing mouse oviduct during that period after birth when ciliogenesis is at a maximum (Staprans, I., and Dirksen, E. R. (1974) J. Cell Biol., 62, 164). To determine the degree to which de novo synthesis and tubulin pools contribute to this increase, [³H]leucine-incorporation experiments were performed in vivo and in culture. Soluble, particulate and axonemal fractions, obtained from homogenized oviducts of 3-, 5-, 8- and 12-day-old suckling mice, were electrophoresed on sodium dodecyl sulfate gels and the specific activity of the tubulin band determined. The present work shows that more than 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts is synthesized de novo. From both the in vivo and in culture experiments we conclude that although tubulin pools are present in mouse oviduct, they are continuously being replenished by newly synthesized protein as there is a rapid outflow from the soluble and particulate to the axonemal fraction into structures such as basal bodies and cilia. This burst of de novo tubulin synthesis corresponds to evidence from electron microscopic autoradiography, where label is present to a greater extent over centriole precursors and basal bodies than over other cell organelles. [³H]leucine incorporation into tubulin was inhibited by cycloheximide, demonstrating that we are dealing with synthesis, while colchicine below 10^?3, M concentration had no effect on tubulin assembly into axonemes.     

Rapid colorimetric assay for intestinal peptide hydrolases

A simple two-step method is described for quantitating the release of free l-phenylalanine, l-leucine, l-methionine, or l-isoleucine from di- or polypeptides. The colorimetric assay is based on the ability of l-amino acid oxidase to catalyze the oxidation of free l-amino acid, but not of peptides. The potent metal chelator 1,10-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.     

The mixed lymphocyte reaction: selective activation and inactivation of the stimulating cells.

Allogeneic cells pretreated for 48 hr with 2 × 10^?6M ouabain have lost the capacity to show the mixed lymphocyte reaction (MLR). Analysis of various combinations of cells in the one-way MLR revealed that this effect was on the stimulating cells and not on the responding cells. Pretreatment of cells from both donors with 10^?7M ouabain caused no change in incorporation of labeled thymidine into DNA during the first 5 days of mixed lymphocyte culture; thereafter, as incorporation by the controls declined, that of the pretreated cells continued to increase. This effect was also on the stimulating cells and not on the responders. The irreversible effects of ouabain are thus either to activate or inactivate the stimulating cells depending on the concentration of the drug; there is little or no effect on the responding cells.     

Kinetic properties of exchangeable calcium in guinea-pig heart mitochondria measured at low concentrations of free calcium and in the presence of Mg2+, ATP4− and inorganic phosphate

The inetic properties of exchangeable Ca²⁺ in isolated guinea-pig heart mitochondria were studied at 25°C in the presence of 0.9 mM free Mg²⁺, ATP, phosphate ions and 0.4 – 0.5 μM free Ca²⁺ using a ⁴⁵Ca²⁺ exchange technique. The simplest system which was found to be consistent with the data was one in which two kinetically-distinct compartments of exchangeable Ca²⁺ are present in the mitochondria. In the presence of 6 mM Na and at 0.4 μM free Ca²⁺, the fractional transfer rates for the transport of Ca²⁺ from these compartments were found to be 0.6 and 0.05 min^?1 and the quantities of exchangeable Ca²⁺ 0.04 and 0.2 μmol/g wet wt heart, respectively. The amount of ⁴⁵Ca²⁺ exchanged increased when the concentration of inorganic phosphate was increased, and decreased slightly when the concentration of free Mg²⁺ was increased from 1 mM to 3 mM. The flux of Ca²⁺ across the boundaries of both compartments was inhibited by an increase in the concentration of extramitochondrial Na⁺. The contribution of mitochondrial Ca²⁺ to compartments of kinetically-distinct exchangeable Ca²⁺ observed in intact cardiac muscle is briefly discussed.     

Qualitative patterns of protein synthesis in the preimplantation mouse embryo. II. During release from facultative delayed implantation.

Qualitative patterns of newly synthesized protein were examined in mouse embryos released from facultative delayed implantation either by removal from the uterus with subsequent culture in vitro, or by the administration of progesterone and estradiol 17β (in vivo) to the ovariectomized mother. It was found that the qualitative pattern of protein synthesis during the first 25 hr following release from delay is the same as the pattern obtained from normal blastocysts immediately prior to implantation. These results are discussed in relation to possible mechanisms of activation of delayed blastocysts.     

Simultaneous measurements of testosterone and three 5a -reduced androgens in the venous effluent of immature rat testes in situ

Simultaneous measurements were made by radioimmunoassay of testosterone, 17β-hydroxy-5 a -androstan-3-one (DHT), 5 a -androstane-3 a, 17 β -diol (3a-diol), and 5 a-androstane-3 β, 17β-diol (3β-diol) in the testicular venous plasma (TVP) and peripheral plasma (PP) of 30, 45, and 55 day old rats. At 30 days of age, the preponderant androgen in both plasmas was 3a-diol but testosterone predominated by day 55. Testosterone levels increased with age in both TVP (6.39, 15.08, and 54.93 ng/ml on days 30, 45, and 55 respectively) and PP (0.13, 0.56, and 1.02 ng/ml on days 30, 45 and 55 respectively) whereas 3a-diol concentrations decreased in TVP (48.07 ng/ml, day 30; 24.85 ng/ml, day 55) though not peripherally (range: 0.41–0.52 ng/ml). DHT was low in both TVP and PP and appeared to rise only slightly although the increase was not statistically significant. Levels of 3β-diol remained low and unchanged. These observations suggest that the total androgen content of the venous effluent from the prepubertal rat test is is quite high and that significant changes in peripheral interconversions of androgens are occurring during sexual maturation.     

Measurement of gamma-aminobutyric acid (GABA) in blood.

Blood GABA levels can be readily determined using a radioreceptor assay or gas chromatography-mass spectrometry. After withdrawal of blood, GABA levels remain stable with 25–50% of the GABA in whole blood found in the plasma fraction. Whole blood GABA concentrations range from 500 pmoles/ml to 1200 pmoles/ml in 8 mammalian species with human values being about 900 pmoles/ml. $\text{in vivo}$ administration of aminooxyacetic acid increases both blood and brain GABA levels to a similar extent.     

Circulating plasma testosterone in the XYY male

A population of 6 XYY and 6 XY individuals participated in a study to compare the levels of free circulating testosterone. No significant differences were found in the amounts of free circulating testosterone or total plasma testosterone. The plasma testosterone levels of the twelve subjects were within the normal range of values (596ng/dl ± 232ng/dl) obtained in our laboratory. Estrogen levels were assayed to determine estrogen influence on testosterone levels. Estrogens were not significantly different in the two groups. Plasma estrogens were within the limits of a random normal population (15–55pg/nl).     

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels into antibody-containing agarose: an improved method for evaluation of the number of immunochemical determinants in polypeptides, with reference to spectrin.

A simple method is described for performing crossed immunoelectrophoresis into antibody-containing agarose when the first-dimension gel contains peptides separated by electrophoresis in sodium dodecyl sulfate. Artifacts produced by sodium dodecyl sulfate are avoided by incorporation of Triton X-100 in the agarose layer. Peptides are located by prestaining (before SDS-acrylamide electrophoresis) with the cycloheptylamylose complex of fluorescamine. Injection of ink into prestained peptide bands produces a line extending from the peptide band location to its precipitin arc, thereby allowing unambiguous assignment of arcs to peptides in situations where peptide bands are not widely separated. The utility of this procedure is illustrated for the erythrocyte membrane protein spectrin.     

Effect of cigarette smoke on drug metabolism in vitro.

N-demethylation of aminopyrine and C-hydroxylation of aniline by hepatic microsomal enzymes were measured during $\text{in vitro}$ exposure to cigarette smoke. Metabolism of aminopyrine during smoke exposure was not significantly altered. Metabolism of aniline during smoke exposure was inhibited 70–80% (P < .001) thus indicating that an initial effect of exposure to cigarette smoke is a decreased rate of biotransformation via C-hydroxylation. This finding, coupled with the findings of other investigators who have shown that the delayed effect of exposure to cigarette smoke is induction of hydroxylase activity, suggests that cigarette smoke produces a biphasic alteration in certain hepatic biotransformation processes.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

The calibration and use of a calcium ion-specific electrode for kinetic studies of mitochondrial calcium transport.

A calcium ion-specific electrode has been used to study calcium transport by isolated,hepatic mitochondria. The methodology used requires only a sensitive pH meter operated in the millivolt mode with the electrode. Free calcium ion concentrations may be followed continuously. Using incubation conditions which cause release of intramitochondrial calcium, the calcium electrode system may also be used to determine total. intramitochondrial calcium. Techniques for the calibration of the electrode response are discussed. Free calcium ion concentrations have been calculated from total calcium concentrations and the association constants for the binding species present in the assay medium. The observation that the electrode response is linear to submicromolar concentrations allows calculation of a linear least-squares fit of millivolt reading to computed free calcium ion concentration. A computer program written in BASIC for these computations is included in Appendix material. The half-maximal rate constant for mitochondrial calcium uptake has been found to occur at a free calcium ion concentration of 6.5 μm. The interaction or Hill coefficient for the process is 2.3, indicating positive cooperativity.