Problems encountered in measuring erythrocyte pyrimidine 5'-nucleotidase activity

An improved method of two-dimensional electrophoresis that allows unequilibrated first-dimension gels to be loaded and electrophoresed on second-dimension gels twice the length used in the O'Farrell technique has been developed. Normally, the electrophoresis of unequilibrated first-dimension gels on long second-dimension gels with the resolving gel set at pH 8.8 results in poor resolution of low-molecular-weight proteins. Adjusting the pH of the resolving gel to pH 8.3 maintains the low-molecular-weight proteins in a stacked configuration during their migration through the length of the 10% acrylamide gel. Utilization of a 10 to 20% exponential polyacrylamide gradient in a resolving gel set at pH 8.3 separates these low-molecular-weight proteins with excellent resolution. Electrophoretic resolution of protein spots in resolving gels set at pH 8.8 is not as sharp as in gels set at pH 8.3, and resolution progressively deteriorates in gels set at higher pH values.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

The oxidation of endogenous ascorbic acid during histamine secretion by rat peritoneal mast cells

Endogenous ascorbic acid is oxidized to the free radical species by rat mast cells during histamine secretion. This antioxidant may function as a radical scavenger of Superoxide and other membrane-damaging radicals known to be liberated by this process. The high levels of ascorbic acid in mast cells may, therefore, function to protect the cell membrane from oxidative damage and thereby promote cell survival after an extensive secretory response.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Computer-assisted analysis of photoacoustic spectra of solid and solution state cyanide methemoglobin

A computer-assisted method for analyzing photoacoustic spectra has been developed. Using this analysis, the relative absorption spectrum and either the chromophore concentration or thermal diffusivity characteristic of a sample can be derived from its photoacoustic spectrum. We have demonstrated the accuracy of the method by analyzing photoacoustic spectra of solution and crystalline-phase bovine cyanide methemoglobin. BASIC and FORTRAN routines used to collect and to analyze photoacoustic spectra are described. Photoacoustic spectroscopy can be used in conjunction with the analytical method presented here to obtain accurate absorption spectra from a variety of solid, opaque, and/or turbid samples.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Multiple forms of chlorophyll-protein complexes from a thermophilic cyanobacterium Synechococcus sp

Eight chlorophyll-proteins were resolved from the thylakoid membranes, or digitonin particles, of a thermophilic cyanobacterium Synechococcus sp. by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six chlorophyll-proteins with slower electrophoretic mobilities were shown to be P700-chlorophyll a-protein complexes (CP1), whereas faster-moving proteins (CP2) were related to photosystem 2. Extraction of CP1 complexes from the membranes with different detergent/chlorophyll ratios and reelectrophoresis of extracted CP1 complexes indicated that the chlorophyll-proteins are closely interrelated with each other; any CP1 complex could be transformed to other CP1 complexes with faster electrophoretic mobilities. This, together with the Ferguson plot and the polypeptide composition, showed that six CP1 complexes are different in terms of polypeptide composition, oligomerization, SDS-binding, or conformation of the proteins but represent, in the order of increasing electrophoretic mobility, increasing degree of modification of the native P700-chlorophyll a-protein.     

Kinetic studies on the membrane-bound and the purified coupling factor-ATPase from Rhodopseudomonas sphaeroides

The activity of membrane-bound and purified ATPase (EC 3.6.1.3) was potentiated by several divalent cations. Highest rates of ATP hydrolysis were obtained when the activity was measured with the (cation-ATP)2- complex. Free ATP and free divalent cations in excess were found to be competitive inhibitors to the complex. The apparent Km (complex) values were lower than the Ki values for free ATP indicating that the (cation-ATP)2- complex is bound more tightly to the enzyme than the free ATP. Based on these results, a binding of the complex to the active site at two points is suggested, namely through the ATP and through the cation. Removal of the coupling factor from the membrane apparently caused conformational changes which resulted in a pronounced alteration of the kinetic parameters of ATPase activity. Whereas highest values in chromatophore-bound ATPase activity were observed in the presence of Mg2+, the purified enzyme became even more active in the presence of Ca2+. The Ki values for free ATP decreased upon solubilization of the enzyme. Free Mg2+ in excess was more inhibitory on the purified ATPase than Ca2+, while free Ca2+ in excess was more inhibitory on the membrane-bound enzyme if compared to Mg2+. Ki values for product inhibition by ADP and Pi were determined. Kinetic analyses of photophosphorylation activity revealed that the (cation-ADP)- complex is the functional substrate. The apparent Km values for the complex and for Pi were estimated. Excess of free cations and ADP inhibited competitively the phosphorylation. Ki(ADP), Ki(Ca2+), and Ki(Mg2+) were calculated by Dixon analyses.     

On the mechanism of irreversible thermoinactivation of enzymes and possibilities for reactivation of “irreversibly” inactivated enzymes

A possible mechanism for so-called irreversible thermoinactivation of enzymes is suggested. We postulated that in many cases “irreversibly thermoinactivated enzyme” is an incorrectly folded polypeptide chain which, during a reasonable experimental interval, cannot transform into a correctly folded, thermodynamically stable and catalytically active conformation because of high kinetic barriers (intrinsic steric hindrances). Proceeding from this model, reactivation of “irreversibly” thermoinactivated trypsin (covalently attached to CNBr-activated Sepharose to prevent intermolecular processes such as aggregation or autolysis) was carried out. Thermoinactivated trypsin was first transformed to random coil by reduction of its disulphide bonds in 8M urea solution and then renaturated into a catalytically active form by reoxidation of disulphide bonds in the presence of dithiothreitol and in the absence of the denaturant.     

Soluble relaxation factor from vascular smooth muscle: A myosin light chain phosphatase?

Extracts from bovine aortic muscularis contain a heat labile and ethanol-stable factor which enhances relaxation of chemically skinned fiber bundles from hog carotid artery. Relaxation promoting fractions obtained after several stages of purification are enriched in myosin light chain phosphatase activity and devoid of calmodulin, myosin light chain kinase, and cAMP-dependent protein kinase activity. These findings suggest that the relaxation-promoting factor may be a myosin light chain phosphatase and that the enzyme plays a significant role in modulating relaxation.     

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.     

Immunoregulation in MRL/Mp-lpr/lpr mice: evidence for decreased helper-T-cell and increased suppressor-T-cell function with age

In vivo and in vitro plaque-forming cell (PFC) responsiveness to sheep erythrocytes (SRBC) was used to assess immunoregulatory function in the autoimmune MRL mouse strain. MRL/Mp-lpr/lpr (MRL/l) mice had good primary and secondary IgM and IgG responses in vivo compared to MRL/Mp-+/+ (MRL/n) mice when young, but with age the MRL/l responses declined markedly. In vitro primary SRBC-specific PFC responses in MRL/l mice declined at the same time as in vivo responses, indicating that the in vivo autoimmune environment could not account for cellular dysfunction. When varied mixtures of T and B cells from MRL/l and MRL/n mice were cultured, abnormalities in MRL/l T-cell function became apparent. T-helper-cell (T_H) function declined rapidly with age, beginning by 2 to $\text{2}\text{1}\text{2}$ months of age. T cells from MRL/l mice 2 months of age and older also had increased suppressor activity when cultured with B cells and MRL/n T cells. The degree of suppressor activity increased with age. The correlation of these findings with results of previous studies by others and with autoimmune disease is discussed.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.     

Activation of natural killer cells in vitro by a product of beta-hemolytic streptococci

Streptokinase-streptodornase (SK-SD), a product of Group A and Group C β-hemolytic streptococci, activates natural killer (NK) cells in vitro. This effect appears to occur without the induction of interferon. It is not present in every individual but is reproducible. There appears to be no correlation between NK activation and assays for cellular immunity such as in vivo delayed skin reactions and/or in vitro cell proliferation.     

The reaction of tetranitromethane with human chorionic gonadotropin.

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding.