A sensitive continuous and discontinuous photometric determination of oxygen, carbon dioxide, and carbon monoxide in gases and fluids.

The paper describes a sensitive, rapid, and precise photometric method for the continuous and discontinuous determination of O₂, CO₂, and CO. The method is based on highly specific color reactions: O₂ is determined by its reaction with alkaline catechol + Fe²⁺ yielding intensively colored products, CO₂ is determined by its color reaction with a solution of fuchsin + hydrazine; and CO is determined by its reaction with hemoglobin. The basic experimental equipment is that of the AutoAnalyzer (cf.Wolf, Zander, and Lang, 1976, Anal. Biochem.74, 585), with an additional chamber for the injection of small gas samples in the case of the discontinuous analysis. Continuously analyzing in a standardized gas flow of 1 ml · min^?1 (STPD), the lower limits of the sensitivities are 50 ppm for O₂, 100 ppm for CO₂, and 50 ppm for CO. The discontinuous analysis of the three gases requires the basic experimental equipment plus an airtight chamber. The lower limits of the amounts are 0.1 μl (STPD) for O₂, 0.2 μl for CO₂, and 0.1 μl for CO.     

On the physical properties and mechanism of action of arylsulfate sulfohydrolase II from Aspergillus oryzae.

Sedimentation equilibrium studies on arylsulfate sulfohydrolase II (EC 3.1.6.1) from Aspergillus oryzae under nondissociating conditions have resulted in a revised molecular weight of 94,900 ± 7100. Sedimentation equilibrium and gel electrophoresis data collected in the presence of the dissociating agents, urea and sodium dodecylsulfate demonstrate that the native enzyme is composed of two identical subunits as suggested by previous studies employing an irreversible inhibitor.The pH dependencies of the kinetic parameters V and $\text{V}\text{K}{}_{\mathrm{m}}$ for the enzymic hydrolysis of 4-nitrophenyl sulfate indicate that two groups of pK_a 4.7 and 6.0 control the activity of the enzyme. The product inorganic sulfate was shown to be a linear competitive inhibitor of the enzyme at pH 4.0, implying that it is a last released product along the reaction pathway. Inhibition by the phenol product was not observed. Enzymic hydrolysis of 4-nitrophenyl sulfate in ¹⁸O enriched water revealed that one atom of solvent oxygen is incorporated per molecule of inorganic sulfate, which is consistent with a mechanism featuring sulfur-oxygen bond cleavage. Evidence is presented based on stopped-flow kinetics, partitioning experiments in the presence of amine nucleophiles, and ¹⁸O exchange studies that collectively suggest that the breakdown of a covalent sulfuryl enzyme intermediate probably is not the rate-limiting step along the reaction pathway.The substrate specificity of the enzyme was examined by testing a variety of sulfate and phosphate esters as inhibitors of the hydrolysis of 4-nitrophenyl sulfate. The Cbz-l-Phe-l-Tyrosine-O-sulfate methyl ester serves as a substrate for the enzyme. Apparently substrate activity requires an aromatic sulfate ester whose binding is enhanced by incorporating the aromatic moiety in a hydrophobic matrix.     

Metabolism of zinc and copper in the neonate: Effect of cadmium administration during late gestation in the rat on the zinc and copper metabolism of the newborn

Rats that have been treated with Cd (1.0 mg/kg body wt., i.v.) on the 18th day of gestation give birth to young, the livers of which are low in Zn, but not in Cu. With increasing age after birth the hepatic concentrations of total and thionein-bound Zn in these animals increase rapidly to maxima at about 7 days, approx. 6 days later than in the newborn of normal dams, whereas the liver Cu concentration reaches a higher maximum at an earlier age than in the control neonate. This rapid uptake of Cu into the liver of the newborn of the Cd-treated dam is not accompanied by a concomitant increase in the concentration of soluble thionein-bound Cu.Cadmium-treatment of the dam retards the weight gain of the liver and, at least during the first 6–8 days postpartum, the increase in body wt. of the newborn. When the hepatic concentrations of thionein-bound Zn is expressed relative to liver wt. instead of age, there is no significant difference between the newborn from normal and Cd-treated dams.The Zn concentrations in blood, brain, stomach, duodenum, pancreas, spleen, kidney and muscle of newborn rats either remain constant, or increase only slightly with age after birth and are not affected significantly by the administration of Cd to the dam in late gestation. This treatment, which probably increases the demand for Zn in the newborn, delays the deposition of Zn in bone and causes a reduction in the Zn concentration of the skin. The Cu concentrations in skin and bone, as well as in other organs of the newborn during the first 24 days postpartum, seem to be unaffected by Cd-treatment of the dam.It is suggested that hepatic Zn-thionein has an essential function in the Zn metabolism of the liver, but is unlikely to control the supply of Zn to other organs in the newborn rat.     

Capacity of different cell types to prime in vivo for secondary in vitro cytotoxic T-cell responses against non-major-histocompatibility antigens

The capacity of several types of cell preparations to induce in vivo a state of memory for a secondary in vitro cytotoxic response against non-major-histocompatibility antigen was markedly reduced (on a per cell basis) by uv-irradiation. This indicated that memory induction requires metabolically active stimulator cells. An “adherent cell preparation” (AC) that was enriched for dendritic cells was among the most effective memory-inducing cell populations; but concanavalin A-activated nylon-wool-nonadherent spleen cells (Con A-NWT) or concanavalin A-activated unfractionated spleen cells (Con A-spl) were on the average equally effective. Normal unfractionated spleen cells (spl) or nonactivated nylon-wool-nonadherent cells (NWT) were markedly less effective on a per cell basis. This pattern of stimulatory activity was in line with the relative stimulatory activity of these cell types in primary cytotoxic responses in the presence of interleukin 2 (IL-2) and also in line with the relative capacity to induce IL-2-dependent proliferation in H-2D-incompatible T-cell populations (cf. W. Dröge et al., J. Immunol.132, 2749, 1984). These differences in the immunogenic potential and the requirement for metabolically active stimulator cells suggested that these cells stimulated the CTL system directly and not indirectly through antigen processing cells of the immunized host. Nevertheless, the secondary cytotoxic response after injection of low numbers of Con A-spl into H-2 heterozygous recipients, (BALB/c × BALB/b)F1, or into recipients with recombinant H-2 haplotype (A.J) was only preferentially but not exclusively restricted to the H-2 haplotype of the immunizing cell populations. Restriction was considerably more complete when AC were used for immunization.     

The reaction of Euglena gracilis cytochrome c-552 with nonphysio-logical oxidants and reductants.

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c₂. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c₂.     

The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.     

The guanosine 5'-diphosphate D-mannose: guanosine 5'-diphosphate L-galactose epimerase of Chlorella pyrenoidosa. Chemical synthesis of guanosine 5'-diphosphate L-galactose and further studies of the enzyme and the reaction it catalyzes.

An enzyme from extracts of the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-galactose was further purified. The substrate guanosine 5′-diphosphate l-galactose was made chemically by the morpholidate procedure. An improved method was developed for the synthesis of an intermediate in that process, β-l-galactopyranosyl phosphate, via an orthoester of l-galactose. Various characteristics of the enzyme and the reaction it catalyzes were studied. A new method using gas-liquid chromatography was introduced for following the course of the reaction with unlabeled substrates.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

Characterization of the glucocorticoid-receptor proteins in the Novikoff hepatoma.

The response of brown adipose and liver mitochondria from the cold-acclimated hamster (Mesocricetus auratus) to the synthetic uncoupler 2-azido-4-nitrophenol has been measured. Brown adipose mitochondria are more readily uncoupled than liver mitochondria. Binding of 2-azido-4-nitrophenol to either kind of mitochondria is competitively inhibited by 2,4-dinitrophenol, by palmitic acid, and by the trifluoromethylphenylhydrazone of carbonyl cyanide. Separate experiments indicated that the number of high-affinity binding sites is approximately the same for either kind of mitochondria; hence it was concluded that observed differences in binding are due to dissimilar dissociation constants of the uncoupler. Brown adipose mitochondria bind 2-azido-4-nitrophenol less tightly than liver mitochondria, but this difference is probably due to the effect of residual long-chain fatty acids which cannot readily be removed. A convenient synthesis of 2-azido-4-nitrophenol is described, along with a method for tritiation of the reagent.     

Studies of a serum albumin-liposome complex as a model lipoprotein membrane

Electron microscopy shows that the lipoprotein dispersions formed from the interaction of negatively charged liposomes with bovine serum albumin contain closed, vesicular, multilamellar structures. Discontinuous density gradient studies indicate that the lipoprotein suspensions are vesicles in which bovine serum albumin homogenously associate with lipid.Low angle X-ray diffraction results show that all the systems, positively and negatively charged, with and without protein, have the characteristic lamellar structure observed in biological membranes. The lamellar spacing (bilayer plus water layer) of negatively charged liposomes without bovine serum albumin is 55 Å. The same lamellar separation in the positively charged system is 108 Å. The lamellar spacing corresponding to bilayer, water, and protein for the negatively charged lipoprotein system is 93 Å while that for the positively charged lipoprotein system is 91 Å. These dimensions suggest that a layer of protein one molecule thick is incorporated between the lamellae bound to the surface of the bilayer.Wide angle X-ray diffraction results indicate no major effect of the protein on the 4.1 Å spacing, characteristic of hexagonal packing of the hydrocarbon chains.A classical light scattering technique is to used to show that the lipoprotein systems are osmotically active. The solute permeability exhibited by these lipoprotein systems follows the sequence (glucose < arabinose < malonamide < glycerol). K⁺ diffusion from negatively charged lipoprotein systems is greater than that found for positively charged lipoprotein systems.     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 2. Kinetic studies on the active site

The purified beta-N-acetylglucosaminidase isolated from Turbatrix aceti hydrolyzes both p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides. The enzyme had Km values of 0.28 and 0.23 mM, Vmax values of 104 and 69 mumol min-1 mg protein-1, and activation energies of 11.7 and 9.9 kcal/mol for the two substrates, respectively. Several lines of experimental evidence show that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same molecule at a single catalytic site. Substrate analogs were synthesized in which the acetamido group of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside, and their 1-thio analogs was modified by replacement of the amido-carbonyl oxygen with sulfur. These substrate analogs competitively inhibited both enzymatic activities. Analysis of the inhibition data indicates that a single catalytic site of the enzyme is responsible for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Competition kinetics between the two substrates further confirm the presence of a single active site for both activities. The pH dependence of the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides has been determined. pKe1 and pKe2 values of 4.7 and 5.2, determined from the dependence of log Vmax/Km on pH, suggest that two carboxyl groups are involved in the reaction mechanism. The heats of ionization of the groups further confirm the above results.     

The effect of sodium ion on antinociception and opiate binding in vivo.

Large quantities of NaCl and CaCl₂ but not KCl given intrapertoneally decreased the antinociceptive activity of morphine. NaCl also antagonized the effect of morphine on the stereo-specific binding of opiates. This high dose of NaCl doubled the level of sodium in the brain but did not alter the specific gravity of brain tissue. These $\text{in}$$\text{vivo}$ effects of NaCl confirm the antagonistic effects of NaCl $\text{in}$$\text{vitro}$ that have been reported.     

In situ effector pathways of allograft destruction. 2. Generation of the "humoral" response in the graft and the graft recipient

The frequency of both immunoglobulin (Ig)-synthesizing and Ig-secreting B cells have been analyzed in DA-to-WF rat renal allografts (and in control WF-to-WF autografts). We have correlated the in situ B-cell responses with corresponding events in the central lymphatic system of the recipient. Intracellular IgM- and IgG-containing plasma cells appeared in an allograft (but not in an autograft) very shortly after the transplantation. The numbers of both cell types in situ was approximately equal, the highest numbers of each being found on Day 4 after transplantation. A similar early response was observed in the recipient's spleen, however, very few Ig-synthesizing cells were seen in the blood. Only a fraction of the Ig-synthesizing cells in the allograft were involved in immunoglobulin secretion. Thus, the recovery of IgG- and IgM-secreting cells from an allograft was 10 and 2% of intracellular IgG- and IgM-containing cells, respectively. It appears, therefore, that allograft-infiltrating Ig-synthesizing B cells either die or migrate elsewhere before secreting immunoglobulin. The B-cell response in the graft occurs very early and is disproportionally high when the very low frequency of B lymphocytes in the allograft is considered. The data provide no evidence for inflammatory B cells being an integral part of graft rejection. Indeed, the possibility remains that the inflammatory B-cell response observed during the rejection process represents a meaningless byproduct of the inflammatory response.     

Purification,properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s_{20, w} = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD⁺ binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K_m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.     

A photochemical method for rapid and precise determination of carbon monoxide levels in blood.

A simple and inexpensive flash photolysis apparatus for determination of the level of carbon monoxide saturation of blood samples is described. Saturation with CO is determined by observing the change in light transmission at 432 nm produced on photolysis of bound CO with a light flash. The procedure is highly specific for carbon monoxide, requires less than 5 μl of blood (obtainable from a finger prick), and has a resolution better than 0.1% in saturation. In addition the apparatus does not require frequent calibration.     

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 μM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.     

In situ effector pathways of allograft destruction. 1. Generation of the "cellular" effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.     

Studies on the mechanism of castanospermine inhibition of alpha- and beta-glucosidases

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine alkaloid that was isolated from the Australian plant, Castanospermum australe. This alkaloid was found to be a potent inhibitor of lysosomal alpha- and beta-glucosidases. In this report, the mechanism of inhibition of amyloglucosidase (an exo-1,4-alpha-glucosidase) and almond emulsin beta-glucosidase was examined. Castanospermine proved to be a competitive inhibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the p-nitrophenyl-alpha-D-glucoside. It was also a competitive inhibitor of almond emulsin beta-glucosidase at pH 6.5, but in this case previous studies had shown that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation mixture had a marked effect on the inhibition. Thus, in all cases, castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower pH values. The pK for castanospermine was found to be 6.09, indicating that the alkaloid was probably more active in the unprotonated form. This was also suggested by the fact that the N-oxide of castanospermine, while still a competitive inhibitor, was 50 to 100 times less active than was castanospermine, and its activity was not markedly altered by pH. These results probably explain why castanospermine is a good inhibitor of the glycoprotein processing enzyme, glucosidase I, since this is a neutral enzyme.