Differential response of testicular and ovarian heme oxygenase activity to metal ions

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.     

Enzymatic heme oxygenase activity in soluble extracts of the unicellular red alga, Cyanidium caldarium

Extracts of the phycocyanin-containing unicellular red alga, Cyanidium caldarium, catalyzed enzymatic cleavage of the heme macrocycle to form the linear tetrapyrrole bilin structure. This is the key first step in the branch of the tetrapyrrole biosynthetic pathway leading to phycobilin photosynthetic accessory pigments. A mixed-function oxidase mechanism, similar to the biliverdin-forming reaction catalyzed by animal cell-derived microsomal heme oxygenase, was indicated by requirements for O2 and a reduced pyridine nucleotide. To avoid enzymatic conversion of the bilin product to phycocyanobilins and subsequent degradation during incubation, mesoheme IX was substituted for the normal physiological substrate, protoheme IX. Mesobiliverdin IX alpha was identified as the primary incubation product by comparative reverse-phase high-pressure liquid chromatography and absorption spectrophotometry. The enzymatic nature of the reaction was indicated by the requirement for cell extract, absence of activity in boiled cell extract, high specificity for NADPH as cosubstrate, formation of the physiologically relevant IX alpha bilin isomer, and over 75% inhibition by 1 microM Sn-protoporphyrin, which has been reported to be a competitive inhibitor of animal microsomal heme oxygenase. On the other hand, coupled oxidation of mesoheme, catalyzed by ascorbate plus pyridine or myoglobin, yielded a mixture of ring-opening mesobiliverdin IX isomers, was not inhibited by Sn-protoporphyrin, and could not use NADPH as the reductant. Unlike the animal microsomal heme oxygenase, the algal reaction appeared to be catalyzed by a soluble enzyme that was not sedimentable by centrifugation for 1 h at 200,000g. Although NADPH was the preferred reductant, small amounts of activity were obtained with NADH or ascorbate. A portion of the activity was retained after gel filtration of the cell extract to remove low-molecular-weight components. Considerable stimulation of activity, particularly in preparations that had been subjected to gel filtration, was obtained by addition of ascorbate to the incubation mixture containing NADPH. The results indicate that C. caldarium possesses a true heme oxygenase system, with properties somewhat different from that catalyzing heme degradation in animals. Taken together with previous results indicating that biliverdin is a precursor to phycocyanobilin, the results suggest that algal heme oxygenase is a component of the phycobilin biosynthetic pathway.     

Induction by barbiturates of a cytochrome P-450-dependent fatty acid monooxygenase in Bacillus megaterium: relationship between barbiturate structure and inducer activity

In a recent communication (Narhi, L. and Fulco, A.J. [1982] J. Biol. Chem. 257, 2147-2150) we found that a soluble cytochrome P-450-dependent fatty acid monooxygenase isolated from Bacillus megaterium ATCC 14581 could be induced about 28-fold by phenobarbital. We have now examined 19 barbiturates and found that 13 significantly induce the specific monooxygenase activity. Of these, 11 are more active than phenobarbital and three (secobarbital, thiamylal and methohexital) are more than 30 times as active on a molar basis. The dialkyl barbiturates without exception show an excellent correlation between increasing lipophilicity and increasing potency as inducers as do most of the barbiturates containing an aromatic substituent. Nevertheless, it is apparent that certain structural features involving factors other than lipophilicity are also necessary for induction. Our finding that barbiturates can cause the non-substrate induction of a cytochrome P-450-dependent monooxygenase in a prokaryote represents a unique discovery that may provide a relatively simple model for apparently similar induction systems in higher animals.     

Configuration of the light induced electric field in thylakoid and its possible role in the kinetics of the 515 nm absorbance chance

Theoretical calculations of the electric potential were carried out using a model of the thylakoid consisting of a spherical dielectric membrane surrounded both inside and outside by highly conductive material. The calculations yielded typical configurations and intensities of the electrical field induced by charges either localized in the membrane or delocalized in the conductive phases. It is shown that the build-up of the uniform transmembrane field is strictly correlated with translocation of charges from the membrane onto the boundaries of the conductive phases which induces a considerable increment in the field-intensity over the greater part of the thylakoid. This shows that the slow rise of the electrochromic absorbance change may be physically related to the slow translocation of charges from the membrane into the conductive phases which is linked to rate-limiting electron transport processes.     

Role of monoamine oxidase-B in medroxyprogesterone acetate (17-acetoxy-6 alpha-methyl-4-pregnene-3, 20-dione) induced changes in brain dopamine levels of rats

The effect of medroxyprogesterone acetate (MPA) on brain monoamine levels and monoamine oxidase (MAO) activity was studied in adult, healthy, non-pregnant female rats. MpA was injected in a single dose of 100 mg/kg i.m. Dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT) levels and MAO activity were estimated fluorometrically in rat brian. No change in DA, NA, 5-HT or MAO activity was observed after 7 days of MPA treatment while a significant decrease in DA levels along with a significant increase in MAO activity was observed after 21 days of MPA treatment. However, there was no change in NA and 5-HT levels after 21 days of MPA administration. The selective reduction of DA by MPA could be due to an increase in MAO-B activity. MPA does not appear to increase MAO-A activity because neither of the specific substrates (NA and 5-HT) of MAO-A was found to be decreased inspite of the increase in MAO activity as estimated by the kynuramine method. These findings suggest the importance of MAO-B also in DA metabolism in rat brain.     

Long-term inhibition of ovulation by a GnRH-antagonist at low dose level

Ac-D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10-GnRH has been prepared by solid phase synthesis. The peptide was found to completely inhibit ovulation when administered on proestrus day in a dose of 1.5 microgram/rat, s.c. The peptide completely inhibited ovulation for a period corresponding to three to four cycles when administered daily in a dose of 5 micrograms/rat, s.c. and caused 70% inhibition of ovulation in a dose of 3 micrograms/rat.     

Phospholipase A2 activity in rat and human lymphocytes

Partial characterization of phospholipase A from rat and human lymphocytes showed that it was much less active in man than in rat. The use of phosphatidylethanolamine labelled in the 2 position as substrate established that phospholipase A activity was 2 acyl-specific. It was maximal at pH 7.0 to 8.0, totally Ca2+ dependent and inhibited by detergents and Indomethacin.     

The lipid integrity of membranes of guinea pig alveolar macrophages studied by nanosecond fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene: The effect of stimulation by concanavalin A and formyl peptides1

Time-resolved fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene was used to monitor physical changes in the membranes of guinea pig alveolar macrophages following stimulation by N-formyl peptides (either N-formylmethionylphenylalanine (FMP) or N-formyl methionyl leucylphenylalanine (FMLP)) and concanavalin A. The anisotropy of diphenylhexatriene in macrophages showed a dependence on stimulation both in the rate of decay and in the value of anisotropy at infinite time. Subtle differences were observed between the effect of concanavalin A and FMLP on the membrane lipid fluidity as detected by fluorescence anisotropy. Concanavalin A stimulation of macrophages decreased the value of the anisotropy at infinite times in the range of 0–20 °C and increased the value at 25–40 °C; and at all temperatures it decreased the rate of decay of anisotropy. At temperatures below 25 °C, the response to FMLP was similar to concanavalin A, but above 25 °C, FMLP only slightly modified the anisotropy decay profile. Another physical parameter, calcium permeability, was examined because Ca⁺² fluxes are dependent upon membrane properties. The temperature-dependent profiles of concanavalin A and FMP-stimulated ⁴⁵Ca⁺² efflux from alveolar macrophages were similar. The rate and extent of ⁴⁵Ca⁺² efflux increased from 4 to 22 °C, with no further increases observed up to 37 °C. This pattern correlated well with observed changes in membrane fluidity.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.     

The development of granulomatous pulmonary inflammation in rabbits by aerosol challenge: I. Release of plasminogen activator by alveolar macrophages

A rabbit model of hypersensitivity pneumonitis (HP) was employed to evaluate the release of plasminogen activator (PA) as a method for monitoring the degree of pulmonary inflammation. PA release from alveolar macrophages (AM) was shown to coincide with inflammation and was maximal at approximately 2 weeks of aerosol challenge. PA release could also be induced in normal AM by peripheral lymphocytes obtained from sensitized animals after incubation with antigen. Unseparated peripheral blood mononuclear cells from experimental animals also exhibited antigen-induced PA release. These results suggest that the measurement of PA release using several different cell populations can be used to evaluate pulmonary inflammation in HP.     

Immunoregulatory activity of culture-induced suppressor macrophages

Rat splenic cells precultured in vitro for 5 days exhibited marked suppressive activity on the secondary cytotoxic T lymphocyte (CTL) response to a Gross virus-induced lymphoma. Suppressive activity was produced by macrophages (MØ) rather than lymphocytes and as low as 1% MØ content was sufficient to achieve completely inhibited CTL responses. Aspirin, indomethacin, and d,l-6-chloro-2-methylcarbazole-2-acetic acid prevented cultured splenic MØ from exerting their inhibitory effect, thereby suggesting a role for prostaglandins in suppression. Events which occurred within the first 24 to 48 hr of the CTL response were susceptible to the suppressive action of MØ since normal CTL responses were obtained if suppressive MØ were added later than Day 2 or if indomethacin was added within the first 24 to 48 hr of culture. Two processes of lymphocyte activation, namely blast transformation and DNA synthesis, were inhibited in the presence of suppressive MØ. However, suppression of these processes did not result in the loss of CTL progenitor cells since CTL responses that were inhibited in the presence of suppressive MØ proceeded normally following their removal.     

Degradation and covalent cross-linking of glutathione reductase by hemin

Hemin (ferriprotoporphyrin IX-chloride) can mediate the covalent cross-linking and degradation of yeast glutathione reductase. This reaction requires both NADPH and oxygen suggesting the involvement of a reduced oxygen species in the cross-linking and degradation process. During the course of the reaction the enzymatic activity of glutathione reductase is rapidly destroyed. Implications of these findings for a regulatory role of hemin in cell biology are discussed.     

Effect of bacitracin on the biodegradation of β-endorphin

β-endorphin was incubated with rat brain homogenate, and the amino acids released were measured by amino acid analysis. Phe, Leu, Tyr, and Lys were liberated in the greatest amount indicating that the cleavage of Leu⁷⁷-Phe⁷⁸ and some Lys-X peptide bonds with endopeptidases followed by the removal of the terminal residues by exopeptidases are the main routes of β-endorphin degradation in the brain. Bacitracin considerably reduced the amino acid release from β-endorphin incubated with rat brain homogenate, and its action is suggested to be due to the inhibition of brain amino- and carboxypeptidases. Bacitracin also potentiated and prolonged the $\text{in vivo}$ analgesic activity of β-endorphin.     

Calmodulin: calcium, potassium, and magnesium ion multiple equilibria and kinetics for interconversion, including the effect of repeated stimulation

Using computer simulations, with realistic values for the intrinsic thermodynamic binding constants and kinetic on and off rate constants at the four metal-binding sites, the calmodulin molecule is shown to have been optimally adjusted by evolution for biological function. Despite a very large number of potential molecular states, depending upon occupation by potassium, calcium, or magnesium ions, the system restricts itself to a small number of physiologically-significant equilibrium species. It appears to possess built-in triggers for biological processes, with rapid (approximately 20 ms time-scale), step-function shaped, changes of the populations of the CMKK, CMMK, CKKK, CMMM (and CCKK) states. The time-response of calmodulin to periodic changes in ionic levels (2 and 5 s-1) makes it quite adequate for repeated stimulations. In short, calmodulin appears as a very selective information transmitter at the molecular level.     

Modification of the surface ATPase activity in cultured hepatoma cells by lipid-depleted media

Several compounds have been described which elute fibronectin from a gelatin-Sepharose affinity support. In the present study, it has been found that the potent chaotrophic agent, lithium di-iodosalicylic acid, is 20-fold more effective in eluting fibronectin from collagen than any other presently described fibronectin elution agent. Lithium di-iodosalicylic acid and certain other fibronectin elution agents have been characterized in regard to several parameters involved in the elution of fibronectin from collagen and plastic substrata. By assaying for retention of the cell adhesive activity of fibronectin, it has been demonstrated that 8 M urea + 0.1 M citric acid, pH 4.7, is the most effective condition for preservation of biological activity following elution of fibronectin from the gelatin-Sepharose affinity support.     

The metabolic defect of methionine dependence occurs frequently in human tumor cell lines

Methionine dependence is the inability of cells to grow when methionine (Met) is replaced by its immediate precursor homocysteine (Hcy) in the culture medium (Met^?Hcy⁺ medium). All normal unestablished cell strains tested to date have been shown to be methionine-independent and thus grow almost as well in Met^?Hcy⁺ medium as they do in Met⁺Hcy^? medium. Results presented here indicate that out of 23 cell lines derived from diverse types of human tumors, 11 do not grow at all in Met^?Hcy⁺ medium and are absolutely methionine-dependent and 3 grow only slightly in this medium. Many of the tumor cell lines tested have little else in common other than the fact that they are methionine-dependent. The high frequency of occurrence of methionine dependence in diverse types of human tumor cells indicates that methionine dependence may be an important aspect of oncogenic transformation and therapeutically exploitable.