Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain. 1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.


SH-proteinase from bean Phaseolus vulgaris var. Perlicka. An SH-proteinase (EC 3.4.22.-) has been isolated from beans of the species Phaseolus vulgaris var. Perlicka. The enzyme is homogeneous when subjected to disc electrophoresis, electrofocusing and sedimentation analysis. The molecular weight was determined as 26,000-28,000 by gel filtration, 30,850 +/- 1500 by sedimentation analysis and 26,930-27,410 by calculation from the amino acid composition (Lys20-21, His3, Arg9, Asp21-22, Thr13, Ser18, Pro12-13, Glu23-24, Gly30, Ala16, Cys/29, Val19, Met1, Ile10, Leu13, Tyr14, Phe6, Trp3). The N-terminal amino acid of the proteinase is isoleucine. The effect of concentration, time of hydrolysis, pH, temperature, cations, anions, urea and guanidine - HCl on the proteolytic activity of the SH-proteinase was studied.


Regulatory responses of arginine deiminase in whole cells of Clostridium sporogenes. Arginine deiminase (EC 3.5.3.6) has been shown to have regulatory properties. The activity was observed to be sigmoidal with respect to substrate concentrations. Addition of histidine to the system caused the abolition of sigmoidal responses. The regulatory properties of the enzyme as well as the desensitising action of histidine could also be demonstrated with whole cell suspensions. The pH of the system also seemed to influence modulations in the enzyme.


Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication. The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.


Conformational studies on parvalbumins by circular dichroism. Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.


EPR studies on the anaerobic reduction of fungal laccase. Evidence for participation of type 2 copper in the reduction mechanism. 1. In anaerobic reduction studies on fungal laccase B (p-diphenol:O2 oxidoreductase, EC 1.14.18.1) with the EPR and stopped-flow techniques it was found that the type 2 copper of the enzyme is rapidly undergoing a reduction-oxidation cycle which is followed by a slower reduction in a couple of seconds. An intermediate EPR signal of unknown origin is formed in the same time-range as the initial reduction of type 2 copper and disappears again when this copper ion is reoxidized. 2. The rate of the anaerobic reoxidation of type 2 copper is similar to the reduction rate of the two-electron acceptor, suggesting that they are interacting in the electron transfer of the enzyme. 3. The changes in the reaction rates of both type 2 and type 3 copper appear to be affected in a similar way by changes in pH. 4. The EPR signal of the type 2 Cu2+ suggests that this ion is liganded to one or more nitrogens.


Involvement of tyrosine residues in the protomer-protomer interaction of Proteus mirabilis flagella as studied by spectroscopic methods, chemical modification and aggregation experiments. Using spectrophotometrical titration, chemical modification, and ultraviolet difference spectral methods, the existence of at least two distinct tyrosine groups in the isolated flagellin of Proteus mirabilis flagella has been established. Three of the five flagellin tyrosines are buried in the protein matrix, whereas the other two seem to lie on the protein surface accessible to perturbants. Also about two tyrosine residues, presumably the latter ones exposed to the environment, can be nitrated with tetranitromethane in the monomeric flagellin with a concomitant loss of the polymerization ability after about one tyrosine per mol flagellin has been nitrated. Nitrated flagellin, homogeneous with respect to molecular weight, degree of nitration and isoelectric point, could be isolated and characterized. On the other hand, it could be shown that in the polymeric flagellum the phenolic groups of all five tyrosine residues are inaccessible to perturbing and modifying reagents. It seems, therefore, that the integrity of the phenolic groups is necessary for the proper folding and aggregation of the flagellin subunits to form the stable helical flagella.


The tyrosyl residues in creatine kinase. Modification by iodine. The effect of the iodination of tyrosyl residues in creatine kinase from rabbit muscle has been investigated at alkaline pH after reversible masking of the reactive thiol groups. The conversion of 4-5 tyrosyl residues to monoiodotyrosines as measured by spectrotitration and by radioactive iodine labelling resulted in almost total loss of enzymic activity. The modified enzyme was unable to bind its nucleotide substrates but no significant conformational change was revealed by optical rotatory dispersion or Stokes radius measurements. However, change in the reactivity of some non-essential thiol groups, presumably those located near the active thiol groups, was observed.


Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups. Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.


Variations of lipid-protein interactions in erythrocyte ghosts as a function of temperature and pH in physiological and non-physiological ranges. A study using a paramagnetic quenching of protein fluorescence by nitroxide lipid analogues. 1. Incorporation of stearic acid nitroxides into erythrocyte ghosts markedly depresses the fluorescence of membrane protein tryptophan residues. 5-Nitroxide stearate quenches fluorescence more efficiently than 16-nitroxide stearate. Both compounds exhibit dynamic (diffusion-limited) quenching above 0.28 mumol/mg protein and static quenching at lower nitroxide protein ratios. Static quenching can be attributed to high affinity binding of nitroxide stearates by membrane protein. The dynamic phase represents distribution of the stearate analogues into a fluid lipid system. 2. Protein fluorophores accessible to quenching by a cholesterol analogue, androstane nitroxide, are saturated at low nitroxide/protein ratios (less than 0.14 mumol/mg protein), without resolution of a static quenching phase. This suggests that sterols are segregated away from protein, probably in CLusters". 3. Paramagnetic quenching by stearate nitroxides increases abruptly between 35 and 50 degrees C. This discontinuous enhancement of quenching by temperature is reversible up to 41 degrees C but irreversible at higher temperatures. The discontinuity is also diminished by lowering pH from 7.3 through 6.5 to 6.0. Quenching by androstane nitroxide increases linearly with temperature up to approx. 41 degrees C and then rises exponentially. We attribute the reversible quenching thermotropism detected by stearate derivatives to reversible, thermotropic unfolding and/or depolymerisation of membrane proteins. The irreversible phase, detected also by the sterol derivative can be attributed to non-reversible protein denaturation. 4. Paramagnetic quenching of membrane tryptophan fluorescence by stearate derivatives is minimal at approx. pH 7.1 (35 degrees C) and increases sharply at lower and higher pH values, suggesting that two categories of protein residues, titrating between pH 6 and 8, profoundly influence the association of fatty acyl chains and penetrating protein segments. Quenching by androstane nitroxide exhibits no significant variation between pH 6 and 8, consistent with other data indicating that erythrocyte membrane sterols are segregated from membrane proteins, probably in clusters. 5. Our new approach confirms previous suggestions of a boundary layer of lipid in close association with some proteins in erythrocyte membranes, as well as experiments indicating that the lipid status in this boundary layer depends on that state of membrane proteins. However, sterols appear to be largely excluded from this boundary domain. Our data further show that lipid-protein interactions in erythrocyte membranes can vary significantly with fluctuations of temperature and pH in the physiological range.


Effect of ions on phospholipid layer structure as indicated by Raman spectroscopy. Various anions and cations are found to induce changes in the layered structure of phosphatidylcholine-water systems as indicated by Raman Spectroscopy. From the ratio of Raman intensities, I1064/I1089, it is inferred that dipositive ions decrease the proportion of gauche character in the hydrocarbon chains, with the relative influence being: Ba2+ less than Mg2+ less than Ca2+ similar to Cd2+. Unipositive ions (Li+, K+ and Na+) produce no observed changes in the Raman spectrum of the lecithin dispersion. The proportion of gauche character of the hydrocarbon chains is found to be nearly independent of the anion for: Br-, Cl-, acetate-, I-, ClO4-, CNS- and SO42-. Dispersions prepared with a solution of KI+I2 produced Raman spectra in which the 1089cm-1 peak, which is characteristic of random lipid chains, was greatly intensified, presumably because of the presence of I3- which is known to penetrate the lipid lamellae. The observed trends are discussed.


Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell. Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H2PO-4 groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.


High molecular weight deoxyribonucleic acid polymerase of LF hepatoma. Purification and properties. A high molecular weight DNA polymerase has been purified from the cytosol of a fast growing hepatoma: LF hepatoma. This enzyme sediments at 11.3 S under polymerization reaction conditions (6 mM KCl) and at 8.3 S in higher salt concentrations (200 mM KCl). In either case, no activity is seen in the 3 to 4 S region where low molecular weight DNA polymerase is found. The purified enzyme has a neutral pH optimum and requires a divalent cation, all four deoxyribonucleoside triphosphates and an initiated DNA template for maximal activity. The synthetic template specificity of LF DNA polymerase has been studied. Although this enzyme cannot copy a polyribonucleotide template, the ribostrand of a synthetic hybrid can be used with low efficiency as an initiator for the synthesis of the complementary deoxyribonucleotide strand. The activity of the purified enzyme is strongly inhibited by thiol-blocking agents. The general properties of LF DNA polymerase are similar to those of high molecular weight mammalian DNA polymerases. In our experimental conditions, the error frequency of this tumoral DNA polymerase was no greater than that  made by the purified high molecular weight DNA polymerase of regenerating rat liver.


Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques. The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.


[Analysis of ribosomes by polyacrylamide gel electrophoresis (author's transl)]. Ribosomal polymers, monomers and subunits from several eukaryotes and prokaryotes were isolated and analyzed by polyacrylamide gel electrophoresis. Extraction of RNA from ribosomal particles after their migration in a polyacrylamide gel, analyses by sedimentation in sucrose gradients and observations in the electron microscope were carried out in parallel. Attention was directed to the reproducibility, the precision and the limitations of the electrophoresis technique.


Effect of plasma lipoproteins and lecithin-cholesterol dispersions on the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of isolated rat hepatocytes. Incubation of rat hepatocytes for 3 h in a medium containing amino acids, salts and albumin resulted in a 2-fold increase in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Inclusion of 10% human plasma, rat serum or dialysed rat serum in the medium resulted in an approximate 7-fold increase in reductase activity. These increases were specific since there was little change in the rate of fatty acid synthesis or in the activity of tyrosine aminotransferase. Reductase levels were increased above control values when high density lipoproteins or leithin dispersions were added to the cells. Lecithin dispersions were also shown to increase the rate of efflux of cell cholesterol to the medium. In contrast, reductase levels were reduced when cells were incubated with low density lipoproteins or cholesterol added as an equimolar cholesterol-lecithin dispersion. This inhibition of the reductase by cholesterol dispersions was dependent on the continued de novo protein synthesis. Our data indicate that in normal rat hepatocytes the relative rates of efflux and influx of cholesterol may be critical to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity and cholesterogenesis.


Phospholipase C from Clostridium novyi type A. I. 1. Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography. 2. The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine. This preparation was free from protease, lipase and oxygen-labile delta-hemolysin. 3. Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates. 4. Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme. 5. This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine.


Factors effecting the solubilisation of stearoyl-coA desaturase of hen liver microsomes. 1. The lipid requirement for maximum desaturase activity was investigated using acetone/water mixtures. It was shown that for maximum stearoyl-CoA desaturase activity of hen liver microsomes neither the total neutral lipid fraction nor 44% of the phospholipid fraction were required. 2. The effect of sodium deoxycholate, Triton X-100, Nonidet P-40 and Bio-solv on the enzyme activity indicated that the neutral detergents had a milder effect than the ionic detergent but both classes could cause considerable irreversible loss of activity. 3. The treatment of the microsomes with 2.5% (v/v) water in acetone greatly improved the effective solubilising power of Triton X-100. The yield of desaturase in the 100 000 X g supernatant obtained by treating the microsomal fraction in this way was strongly dependent upon protein concentration. Maximum solubilisation was achieved with25 mg protein per ml 1% (w/v) Triton X-100 in 0.1 M potassium phosphate buffer pH 7.4. 4. A comparison of the properties of the solubilised and membrane-bound enzyme was made by an investigation of: (i) the temperature and pH optimum, (ii) activation energy and (iii) the effect of inhibitors on the enzyme activity.


Isolation of peroxisomes from the dog kidney cortex. The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.


Hydrolysis of nucleoside phosphates: IV. The metal ion-nucleic base interaction in the Cu2+-promoted dephosphorylation of the 5'-di- and 5'-triphosphates of cytidine, inosine and guanosine, and their protection toward hydrolysis by coordination to Cu(2,2'-bipyridyl)2+. The dephosphorylation of CTP, GTP, ITP, ATP, CDP, GDP, IDP and ADP was characterized by measuring the first-order rate constant (50 degrees; I = 0.1, NaClO4) in dependence on pH (2 to 10). Except with CTP and CDP, the reactions are significantly accelerated by Cu2+ and pass through pH optima. By computing the pH dependence of the distribution of the several species present in the nucleotide (NP) systems, it is shown that the most reactive species is Cu(NP). Cu(NP-H), where N(1) is deprotonated, is somewhat less reactive. In both types of complexes, a metal ion-nucleic base interaction, which is responsible for the increased reactivity, occurs, i.e., macrochelates involving the phosphate chains and the base moieties are formed. In accord herewith, CTP and CDP are rather stable as the coordination tendency of the cytosine moiety is small. Furthermore, in the ternary complexes Cu(2,2'-bipyridyl)(NP) and Cu-(2,2'-bipyridyl)(NP-H), where the formation of a macrochelate is inhibited, the nucleotides are protected. The structure-reactivity relationship is also evident with Cu(ITP)2- and Cu(IDP)- which exist only in part as macrochelates; hence, they are less reactive than for example Cu(ATP)2- or Cu(ADP)-. With the aid of the initial rate, vo = d[PO4(3-)]/dt, the rate laws of the ascending side of the pH optima were determined: vo = k[Cu(NP)]/[H+]. A reaction mechanism that includes an intermolecular attack of OH- at the terminal phosphate group is proposed. The descending side of the pH optimum is attributed to the formation of CU(NP)(OH) or Cu(NP-H)(OH), where the Cu2+-base interaction is insignificant. However, these hydroxy complexes are still somewhat faster dephosphorylated than the free nucleotides. This is attributed to an intramolecular attack of the bound OH- at the terminal phosphate group.


Occurrence and properties of fetal intestinal glycosidases (disaccharidases) in human amniotic fluid. alpha-Glucosidase, beta-glucosidase and beta-galactosidase were studied in cell-free amniotic fluid samples using corresponding 4-methylumbelliferyl-glycosides and a series of disaccharides (maltose, sucrose, trehalose, turanose, cellobiose, gentiobiose and lactose) as substrates. The glycosidases exhibited several properties of intestinal disaccharidases such as pH optimum between 5.2 and 6.4, more activity towards the disaccharides than the artificial substrates, tight association of the activities with sedimentable complexes and beta-glucosidase and beta-galactosidase activities exerted by a single catalytic site. With the disaccharides as substrates, the amniotic fluid glycosidase activities were well correlated to those reported in the literature for fetal intestine of corresponding gestational ages. The presence of intestinal disaccharidases in amniotic fluid indicates that the fetal intestine contributes to the protein and enzymes of amniotic fluid.


Glutamine synthetase and glutamyltransferase in developing chick and rat tissues. Concomitant determination of gamma-glutamine-hydroxylamine-glutamyltransferase (GT) and gamma-glutamyl hydroxamate synthetase (GS) activities in chick retina, brain and liver between the 13th day of incubation and a day after hatching showed that while both activities increased late in incubation, in neural tissues their rises were not simultaneous throughout development; in liver both activities were already high on the 14th day of incubation and changed in parallel thereafter. GS and GT activities could be evoked prematurely in all three tissues by cortisol, but GT activity showed higher responses to the hormone. GT and GS were separable by differential solubilization in chick liver but not in retina of chick or rat. The wide spread of the ratios of GT:GS activities in homogenates of a number of chick and rat tissues (1:1 to 73:1) indicates that the GS reaction is not catalyzed by the same protein that catalyzes the GT reactions. Relative amounts of GT(T) (free of GS activity) and of variants of GS (with different competences to catalyze the GT reaction) may govern the changes between the two activities during development and their distribution among different adult tissues in chick and rat.


Non-ionic diffusion of pentobarbital across human chorion laeve. Diffusion of the weak organic acid, pentobarbital, across human chorion laeve in vitro shows an inverse correlation with pH, when corrected for extracellular space permeation by the concurrent measurement of membrane permeability to the lipid insoluble molecule, p-aminohippurate. The temperature dependence of pentobarbital diffusion at different pHs supports the concept that the additional net flux of solute at lower pH occurs by transcellular permeation by the non-ionized moiety, the observed increase corresponding roughly to the dissociation curve for pentobarbital.


Interaction of mescaline with phenothiazines: effect on behavior, body temperature, and tissue levels of hallucinogen in mice. Mescaline (25 mg/kg; 66 muc/kg) was injected (ip) in mice 45 min before chlorpromazine (CPZ, 2.5, 5, 15 mg/kg), thioridazine (10, 30, 45 mg/kg), or chlorpromazine-sulfoxide (CPZ-SO, 15 mg/kg). Excitement, agitation, slight increase in ventilation and occasional head-shaking were seen 30 min after mescaline and continued for 30-45 min thereafter; locomotor activity and the number of scratching events were significantly increased during this period. CPZ (2.5, 5, 15 mg/kg) and thioridazine (10, 30, 45 mg/kg) partially or completely blocked mescaline-induced gross behavior; CPZ-SO (15 mg/kg) was not effective. Increased scratching responses and locomotor activity induced by mescaline were antagonized by all doses of CPZ and thioridazine; at higher doses, both CPZ (7.5, 15 mg/kg) and thioridazine (45 mg/kg) induced cataleptic-like condition and marked hypothermia. Tissue levels of mescaline, examined 3 hr after its administration, were increased by all doses of CPZ and a higher dose of thioridazine (45 mg/kg); CPZ-SO and lower doses of thioridazine had no effect.


Monitoring in newborn intensive care.

Monitoring in newborn intensive care. The major difference between adult and newborn intensive care is the importance in the latter of respiratory problems requiring extensive surveillance and support. Impaired gas exchange in the lung, together with disturbed breathing patterns, often including the temporary cessation of breathing (apnoea), are characteristic of respiratory distress in pre-term infants. New apnoea monitors continue to be introduced, and the recent development of means for the continuous measurement of arterial oxygen in the newborn, thus allowing dangerously high and low oxygen levels to be avoided, must represent a significant advance in both physiological measurement and neonatal intensive care.


Hydrolysis of particulate tributyrin in a fluidized lipase reactor. Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis-Menten kinetics were calculated. The activity of the stainless steel-lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel-lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme-substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contact the immobilized enzyme is hydrolyzed to a significant extent. The experimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.


Preparation and kinetic properties of gel entrapped urate oxidase. Urate oxidase from hog liver (urate: oxygen oxidoreductase, EC 1.7.33) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 47% retention of activity. The kinetic behavior of the gel entrapped enzyme has been studied in a slurried tank reactor using uric acid as substrate. Internal diffusion effects were found to be negligible for particle sizes below 128 mum. A threefold increase in Km (app) was observed for the 128 mum particles and attributed to diffusional effects. The pH activity profile of the gel entrapped enzyme was bell-shaped at high substrate concentration and could be fitted to a titration curve of two ionizable groups, a basic group having a pK of 7.9 and an acidic group with a pK of 11.0. The gel entrapped enzyme showed excellent stability between pH 6.5 and 10.5.


The response of a bioelectrochemical cell with Saccharomyces cerevisiae metabolizing glucose under various fermentation conditions. Working conditions of a biochemical fuel cell formed by an oxygen cathode and a platinum bioanode in a Saccharomyces cerevisiae suspension metabolizing glucose are described. The biocell response in terms of bioanode potential and current drainage under different fermentation conditions is reported. A kinetic equation relating the current, the number of microorganisms, and the substrate concentration is obtained. The bioanode potential corresponds to that of an oxygen concentration polarization cell.


[Specificity of tyrosine metabolism depending on the state of melaninogenesis]. The excretion of metabolites of tyrosine (p-hydroxypyruvic acid-p-HPA, homogentisinic acid-HGA, total keto acids-TKA) and the activity of tyrosine aminotransferase of the tissues of 36 albino and 36 black rabbits was measured. The initial level of tyrosine metabolites in the urine of black and albino rabbits differed but little from one another. With the introduction of L-tyrosine, the quantity of the excreted p-HPA increased sharply, and of the HGA decreased in the albino rabbits. Among black rabbits an increase of the HGA excretion with a comparatively stable level of the excreted p-HPA was noted. Among all the tissues investigated only in the skin and the liver of albino rabbits there was a sharp increase in the initial tyrosine aminotransferase activity after the feeding of L-tyrosine, which testified to a probable adaptive synthesis of the enzyme. Analysis of the data obtained showed that tyrosine metabolism probably depended on the state of melaninogenesis.


An electrophysiological analysis of the effects of amine-uptake blockers and alpha-adrenoceptor blockers on adrenergic neuromuscular transmission. 1 An electrophysiological study has been made of the effects of either blocking noradrenaline (NA) uptake or alpha-adrenoceptors on conduction in adrenergic preterminal axons and on NA release. 2 The excitatory junction potential (e.j.p.) evoked by a single stimulus increased slightly in duration (maximum 20%) in the presence of high concentrations of desipramine or cocaine (larger than or equal to 1 mug/ml) but there was no change in the spontaneous miniature excitatory junction potential (m.e.j.p.s); the single compound preterminal action potential was decreased in amplitude by a maximum of 10%. The e.j.p., m.e.j.p. and the terminal action potential were not altered by lower concentrations of these drugs (less than mug/ml). 3 The increased decline of the e.j.p. amplitude observed during the first few hundred impulses at high frequencies (10 Hz) in the presence of desipramine or cocaine was accompanied by a similar decline in the amplitude of the preterminal compound action potential, suggesting that the latter gave rise to the former. 4 These observations suggest that the action on post-synaptic alpha-adrenoceptors of NA released by single impulses is terminated by diffusion, and that any NA which is subsequently taken up into nerves is metabolized. 5 All the alpha-adrenoceptor blocking drugs tested reversed the normal depression in e.j.p. amplitude observed during the first few hundred impulses at high frequencies to facilitation; this was unaccompanied by any changes in the preterminal compound action potential. 6 Alpha-Adrenoceptor blocking drugs did not alter the potentiating effect which a conditioning impulse had on the amplitude of the e.j.p. evoked by a subsequent test impulse. The facilitated release of NA during trains of impulses was quantitatively predicted in terms of the addition of the individual potentiations introduced by each impulse in the train. 7 It is suggested that if there is an auto-inhibition of NA release, then it is unlikely that the pre- and post-synaptic alpha-adrenoceptors are identical.


Epidemiology of cleft lip and palate. An attempt to rule out chance correlations. The findings of a previous epidemiological study on oral clefts (599 children) were tested in an independent sample of 194 children, using the same source as for the previous study (Finnish Register of Congenital Malformations). Several of the earlier results--such as seasonal variation and associations between clefts and parental age, social factors, and emotional stress--were not reproduced. It is concluded that chance correlations introduce a marked problem to epidemiological studies. The findings of both studies show that there is an exceptionally high incidence of cleft palate in Finland. Significant geographical variations and associations between clefts and prematurity, threatened abortion during the first and second trimesters, maternal drug consumption during the first trimester, and influenza and fever during the first trimester were found. The possible role of these findings in the aetiology of oral clefts is discussed, and particular attention is paid to the possible teratogenicity of salicylates.


Preparation and monitoring of marijuana smoke condensate samples. The objective of this study was the production of smoke condensate from the marijuana cigarettes on schedule for the bioassay study and involved quality control on each batch of smoke condensate. Examination of the condensate production data (tables 1 and 2) reveal that it is not possible to strictly control the butt length of cigarettes when smoked on a robot smoking machine thereby accounting for some variation in the yield of the dry particulate matter and increment9-tetrahydrocannabinol. No increment8-tetrahydrocannabinol was detected in any batch of the smoke condensate. Recently, Hoffman and co-workers (13) have reported that marijuana that exhibited carcinogenic potential similar to tobacco cigarette smoke condensate in the dermal assay in mice.


Symptomatic treatment of agoraphobia and social phobias: a follow-up study. 1. Twenty-six out of 28 out-patients with agoraphobia and social phobias who had originally been treated with phenelzine or placebo in a double-blind clinical trial were followed up for a mean period of one year. During the follow-up period patients received further pharmacotherapy or behaviour therapy, except that those patients originally receiving placebo were not allowed therapy with monoamine oxidase inhibitors. 2. Ratings of phobic and additional symptoms, social adjustment and degree of personality disorder were made after one year by one of the authors (D.S.) who had no prior knowledge of the treatment each patient had received. 3. There were no significant differences in any of the ratings between the patients of the two groups, but those originally receiving placebo had more additional treatment in the follow-up period. Patients continuing to receive phenelzine frequently experienced a return of symptoms if the drug was withdrawn before six months treatment had elapsed. 4. Degree of personality disorder showed a significant negative correlation (p = -0.6) with improvement in the phenelzine group but not in those receiving placebo originally. 5. Improvement in social adjustment items was less than improvement in symptoms at follow-up. The implications of this are discussed. 6. The results suggest that phenelzine is of comparable efficacy to other symptomatic treatments for agoraphobia and social phobias, but that it acts mainly by symptom suppression. Prolonged treatment in patients with personality disorders is not indicated, for improvement is less likely and the dangers of dependence are greater.


Treatment of chronic obsessive-compulsive neurosis by in-vivo exposure. A two-year follow-up and issues in treatment. Twenty patients with chronic obsessive-compulsive rituals were treated in a partially controlled design by in-vivo (real life) exposure with self-imposed response prevention. Treatment included 4-12 weeks as in-patients, and lasted a mean of 23 sessions. All patients were followed-up for at least two years. No patients dropped out during the trial, though one refused domiciliary treatment after discharge. Significant improvement in compulsions was found after three weeks of real-life exposure, and continued during follow-up. At two years follow-up 14 patients were much improved, one improved and 5 unchanged; in a third year of follow-up the improved patient became symptom-free after further exposure treatment. Improvement after three weeks exposure predicted good outcome at 6 and 12 months follow-up. Muscular relaxation treatment had no significant effect on rituals. Modelling of exposure conferred no advantage over exposure alone for the group as a whole, though it may help selected patients. The role of response prevention is unknown. Patients' commitment to treatment facilitates exposure. Domiciliary treatment with involvement of family members in therapy seems crucial in some cases. Pilot group treatment of patients and families together suggests that this may be a useful adjuvant to individual treatment by increasing motivation and aiding follow-up. Compulsive slowness presents special treatment problems but can be improved by a prompting and pacing approach. The course of rituals was often independent of that of agoraphobia, marital problems and depression where these had initially coexisted with rituals. Depressive episodes were common before, during and after treatment, and required tricyclic medication. The trial sample was predominantly female but was otherwise typical of patients with compulsive rituals. Of the 125 obsessive-compulsives seen in the first author's unit over four years 96 per cent were offered behavioural or anti-depressant treatment. One quarter refused behavioural treatment after it was offered. Real-life exposure with self-imposed response prevention is usually an effective procedure for lasting reduction of chronic compulsive rituals in well motivated patients.


The effect of an antibacterial drug on urinary calcium, magnesium and phosphorus excretion in patients with neurogenic bladder. The effect of the simultaneous administration of hexamine mandelate and methionine on the urinary excretion of calcium, magnesium and phosphate is reported in 3 groups of paraplegic patients. Long-term therapy with G 500 increases bone loss in paralysed patients, and is contra-indicated when there is infection of the urinary tract by urea-splitting organisms.


Properties of gamma-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. The binding of GABA to crude synaptic membrane fractions of the rat central nervous system in the absence of sodium appears to involve synaptic receptor sites for GABA. The sodium-independent GABA receptor binding differs in a number of features from sodium-dependent GABA binding. In fresh tissue sodium-dependent GABA binding is about 10 times greater than sodium-independent binding, while freezing and thawing the brain membranes virtually abolishes sodium-dependent binding but increases sodium-independent GABA receptor binding about 2-fold. Both sodium-dependent and -independent GABA binding are saturable processes with dissociation constants of 1.2 muM and 0.37 muM respectively. Sodium-dependent GABA binding is much more sensitive to extremes of temperature and pH than is sodium-dependent GABA binding. In addition, regional variations in synaptosomal uptake of GABA, sodium-dependent and sodium-independent GABA binding are markedly different. Also, amino acids and drugs differ in their potencies as inhibitors of sodium-dependent and sodium-independent GABA binding, with the relative affinity of several agents for sodium-independent GABA receptor sites closely paralleling their abilities to mimic neurophysiologic effects of GABA at postsynaptic receptors. Though there are some similarities between the influence of drugs and amino acid analogues on sodium-dependent binding and synaptosomal accumulation of GABA there are also marked differences such as a much greater affinity of beta-alanine for the sodium-dependent GABA binding sites than for synaptosomal GABA uptake. This suggests that the sodium-dependent GABA binding may involve uptake sites for the GABA accumulating system of glia rather than for neuronal GABA transport.


Depletion of vesicles and fatigue of transmission at a vertebrate central synapse. Synapses from Mauthner to giant fibers in the hatchetfish are chemically transmitting excitatory axo-axonic synapses located in the medulla. The synapses are 4--10 mum in diameter and easily identified for electron microscopy. Presynaptic vesicles are clustered near the contact regions and are round, clear and 40-60 nm in diameter. Stimulation of the Mauthner fiber at 10/sec for 10 min greatly reduces PSP amplitude and causes profound changes in presynaptic structures. Synaptic vesicles become few in number and there is a marked accumulation of irregular membranous structures. These changes are reversible. During the recovery period, the number of synaptic vesicles progressively increases to control values, and the number of irregular membranous structures declines. Further, stimulation during cooling induces depletion of vesicles together with a great increase in the surface area of the presynaptic membrane and in the number of coated vesicles. Internal irregular membranous structures are few. Our data provide evidence for the vesicular release of transmitter and are consistent with there being a mechanism of membrane recycling in which vesicle membrane fuses with the presynaptic membrane and is reclaimed from it by coated vesicles that then coalesce to form irregular membranous structures from which new synaptic vesicles are formed.


Fatigue and recovery of transmission at the Mauthner fiber-giant fiber synapse of the hatchetfish. When the Mauthner fiber-giant fiber synapse of the hatchetfish is activated at gradually increasing frequencies, postsynaptic potentials (PSPs) in the giant fiber become progressively smaller, but complete failures of transmission are not observed even when PSP size is as small or smaller than miniature PSPs (mPSPs) simultaneously recorded. On the assumption of a Poisson distribution of amplitudes, calculations from the absence of failures and from variance suggest that guantum number remains at least as high as 5--10 and that quantal size is greatly reduced. During tetanic stimulation the frequency of mPSPs first increases and then decreases again, sometimes to a very low frequency. However, mPSP amplitude is reduced by no more than about 50%, which indicates that quanta giving rise to mPSPs come from a different population of vesicles than those comprising evoked PSPs. During rest following a tetanus, calculated quantal size in evoked PSPs recovers within several hundred milliseconds to mPSP size simultaneouly recorded. Most of this recovery time represents time for filling, since vesicles can be supplied at much higher rates during tetanic stimulation. After one second rest PSP amplitude exceeds threshold but recovery for later PSPs in a short train requires many minutes. The slowness of this recovery is consistent with the morphological demonstration of slow recovery of the vesicle population after depletion. These data are interpreted in terms of vesicle release, depletion and membrane recycling. Following depletion new vesicles are released after only partial filling which accounts for small quanta. Very small mPSPs are not seen because filling time is short compared to time for release as mPSPs. Since quantal size can be gradually reduced, release can interrupt filling, and filling and release sites are likely to be the same. The data in combination with the morphological observations support the hypothesis of vesicular release of transmitter and provide new evidence as to rates and sites for filling of vesicles.


Optimal conditions for protein synthesis in incubated slices of rat brain. Optimal conditions for protein synthesis in incubated slices of rat brain were determined to be: thickness 0.3 mm under air (for newborn 0.4-0.6 mm) or 0.5 mm under oxygen; temperature, 35-36 degrees C; hepes (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) buffer; K+, 6-8 mM; Ca2+, 2-3 mM. Though maximum incorporation was found with a Na+ concentration of 110-12- mM. Though maximum incorporation was found with a Na+ concentration of 110-120mM, this requirement appears to be partly osmotic. The Na+ concentration may be reduced to 80 mM without inhibition of incorporation provided adjustment is made for osmotic balance. Mg2+. Optimal pH was 7.2-7.4. The rate of protein synthesis in this medium is 0.08-0.09% replacement of the protein amino acid/h in slices from adults and 1.6%/h in slices from 3-day-old rats. Thus slices from adults synthesize protein at 10-20% of the in vivo rate whereas slices at 3-day-old brainincorporate amino acid at 70-80% of the in vivo rate for young rats.


[Effect of cocarboxylase on hyperlactatemia and hyperpyruvicemia induced by phenformin in dogs]. In the normal anesthetized dog, a cocarboxylase perfusion considerably reduced the increase in blood lactate and pyruvate levels provoked by the intraduodenal injection of phenformin (30 mg/kg); furthermore it seems to counteract the increase of the lactates/pyruvates ratio and opposes the fall in blood pH.


[Effect of repeated alcohol administration on urine secretion and antidiuretic hormone in rats]. The administration of ehtanol by gavage immediately produced a maintened hyperdiuresis, a transient decrease of urinary osmolality and antidiuretic hormone secretion, followed by increased plasmatic and urinary antidiuretic hormone concentrations. Chronic intoxication enhanced these effects probably due to central disturbance.


[Effect of acetazolamide on arterial blood lactate levels during recuperation in air from three days of hypercapnia]. After a three day-period of hypercapnia, carbonic anhydrase blockade by acetazolamide inhibits the development of arterial blood alcalosis during the return in normal environment, and thus eliminates the transient increase in lactacidemia due to glycolysis stimulation.


[Donnan equilibrium and kinetics of uric acid transport across human erythrocyte membrane]. The study of the effects of varying pH and ionic composition of external solution on the uric acid ratio (see article) for human erythrocytes at equilibrium and on its permeability constant shows that the distribution of uric acid agrees with the laws of the Gibbs-Donnan equilibrium and its passage across the human erythrocyte membrane with the law of ionic diffusion.


Evidence for the presence of secondary calcium phosphate in bone and its stabilization by acid production. Calvaria from three 4-day-old chicks were incubated in a variety of buffers to study the parameters controlling the equilibration of bone with its supporting fluid environment. Strong inferential chemical evidence was obtained for the presence in viable bone of some calcium phosphate phase of high solubility apparently governed by the Ksp of CaHPO4-2H2O. In dead bone, this phase underwent spontaneous conversion to a higher Ca/P ratio. In viable bone the soluble phase appeared to be stabilized by the metabolic production of acid (lactate) as revealed by the effects of selective inhibitors.


Preparation and properties of matrix-supported horseradish peroxidase. A new method of enzyme immobilization has been described using poly(4-methacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The enzyme coupling step proceeded through nucleophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer-enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics, and thermal denaturation. Potential uses of the polymerenzyme system in chemical synthesis of benzoquinone derivatives are discussed.


Phosphoenolpyruvate carboxylase of Thiobacillus thioparus. I. General properties. Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxylase (phosphorylating), EC 4.1.1.31) was purified 19-fold from the obligate chemoautotroph, Thiobacillus thioparus. Michaelis constants for the substrates were found to be 0.44 mM for phosphoenolpyruvate, 0.89 mM for bicarbonate, and 0.37 mM for magnesium, using Tris-HC1, pH 7.3. 1-Aspartate, 1-malate, and orthophosphate were found to be inhibitors of enzyme activity, while acetyl CoA, FDP, GTP, and CDP had no effect. Dioxane greatly stimulated enzyme activity.


Kinetic studies on soluble and insoluble urokinases. A water-insoluble urokinase (ins-UK) was prepared by covalent coupling to an electrostatically neutral polyacrylamide derivative. The esteratic activity retained by the bound enzyme is about 70 percent of that of the soluble urokinase (UK). Comparative kinetic studies of these two forms of the enzyme were undertaken on lysine esters: N-alpha-acetyl-L-lysine-methyl ester (ALEe) and N-alpha acetylglycyl-L-lysine methyl ester (AGLMe). It was first observed that these substrates both exhibit a marked inhibitory effect toward soluble UK, whereas this phenomenon was less manifest with the insoluble form of the enzyme. Michaelis constants and maximal velocities measured at 33 degrees C, for UK and ins-UK, were identical when ALMe was used, but slightly different with AGLMe. Determination of initial velocities, at a series of pH values shows only minimal differences in the behavior of the soluble enzyme with respect to that of the insoluble form. However, over a range of temperatures, differing Km values for these two enzyme forms were obtained using AGLMe as the substrate. These last results suggest possible interactions between the substrate and the insoluble carrier of the enzyme.


Ammoniagenesis: d-glutamyltransferase as a source of ammonia in the rat kidney. The contribution of D-glutamyltransferase (D-GT) (EC 2.3.2.1) to total renal ammonia production was determined by employing DL-methionine-DL-sulfoximine (MSO) as an inhibitor of D-GT. Rat kidney homogenates were assayed for NH3-liberating activity under optimal D-GT or gamma-glutamyltranspeptidase (gamma-GTP) (EC 2.3.2.2) conditions. MSO inhibits only D-GT activity. The contribution of D-GT to total renal ammonia production was then evaluated in the isolated perfused rat kidney employing identical substrate (5 mM L-glutamine) and inhibitor (15 mM MSO) concentrations as employed in the homogenate study. Under these conditions, MSO inhibits 70 percent of the total ammonia production by the normal kidney; in addition, the ratio of ammonia produced per glutamine taken up rose from 1.0 to 1.8. In kidneys from chronically acidotic rats, MSO reduced total ammonia production only 35 percent while the NH3/glutamine ratio rose from 1.0 to 1.8. D-GT appears to be the predominant source of NH3 production in the normal rat kidney; gamma-GTP does not contribute significantly. The rise in the NH3/glutamine ratio after D-GT inhibition is consistent with glutamine utilization via the activated mitochondrial glutaminase (EC 3.5.1.2)-glutamate dehydrogenase (EC 1.4.1.2) pathway.


The action of beta-galactosidase (Escherichia coli) on allolactose. The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.


Purification and characterization of a trypsin inhibitor from Solanum tuberosum. A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.


Amino acid transport in a water-mould: the possible regulatory roles of calcium and N6-(delta2-isopentenyl)adenine. Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different systems elucidated are: (i) methionine, (ii) cysteine. (iii) proline, (iv) serine-threonine, (v) aspartic and glutamic  acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine-tyrosine-tryptophan and leucine-isoleucine-valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.


Sequential production of polygalacturonase, cellulase, and pectin lyase by Rhizoctonia solani. The sequence of appearance of cell wall degrading enzymes of Rhizoctonia solani propagules was followed. Polygalacturonase (PG; EC 3.2.1.15) was induced earlier by sodium polypectate (NaPP) as compared with the induction of cellulase (Cx; EC 3.2.1.4) by carboxymethyl cellulose (CMC), cellobiose, or fibrous cellulose powder. Increasing CMC concentration to 0.5% shortened the time of Cx appearance. In Czapek medium containing citrus pectin, pectin lyase (PL; EC 4.2.2.10) was produced faster and at higher amounts than in a medium containing NaPP as the sole carbon source. PG appearance also preceded that of PL in media simultaneously supplemented with their respective inducers. NaPP, which induced production of PG, repressed Cx production. Among the Cx inducers, only CMC and cellobiose repressed PG production to any extent. At pH 6.0, either in a synthetic medium or on autoclaved bean hypocotyl segments, a delay in PG production as compared with Cx and Pl production was observed. Optimal pH levels for enzyme production and activity were 4.0 and 5.0 for PG, and 5.5 for Cx, and 8.0 and 7.5 for PL. PG was less repressed than Cx by glucose, cellobiose, and monogalacturonic acid, while PL was not affected.


Fungitoxicity of 1,4-naphthoquinones to Candida albicans and Trichophyton mentagrophytes. Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.


Proteolytic activity of Oidiodendron kalrai. The physiochemical characteristics of the intracellular proteolytic enzymes of Oidiodendron kalari, a neuropathogenic fungus, were studied. The organism in the yeast phase was grown in a semisynthetic medium containing 1% tryptone, at 37 degrees C for 48 hr, on a gyrotory shaker. The crude extract was prepared by breaking the cells in a French pressure cell and the proteolytci activity was tested against biological substrates. The cell-free extract hydrolyzed casein, hemoglobin, lactalbumin, gelatin, elastin, collagen and purified rabbit renal basement membrane to various degrees. Optimal proteolytic activity was observed at pH 6 and at 32 degrees C. Calcium and EDTA did not affect the enzymatic activity; however, activity was partially inhibited by sulfhydryl-blocking agents and by heat-inactivated horse, calf, and human serum. The extract was totally inactivated by exposure to a temperature of 70 degrees C for 60 min. Storage at -76 degrees C or -15 degrees C for 6 months or at 4 degrees C for 4 weeks did not affect protease activity.


Histochemical characterization of mucosubstances in bone and soft tissue-tumors. The present investigation endeavors to characterize the mucosubstance content of 170 myxoid and chondromatous tumors and chordomas by histochemical methods. The results obtained using the critical electrolyte concentration (CEC) method as introduced by Scott and co-workers23,24 were compared with those obtained by staining with alcian blue and toluidine blue at different pH's with and without pretreatment with bovine testicular hyaluronidase. Tissues known biochemically to contain different heteroglycans were used as controls: synovial fluid and cock's comb (hyaluronic acid) stained with alcian blue up to a MgCl2 concentration of 0.1 M; fetal cartilage (chondroitin 4- and 6-sulphate) pulposus with notochordal remnants (keratan sulphate) up 10 1.0 M. The staining reaction of intramuscular myxoma and myxoid liposarcoma corresponded to that of synovial fluid and cock's comb (containing hyaluronic acid). Benign chondromatous tumors (osteochondroma, enchondroma, extraskeletal chondroma, chondromatosis in bursae, synovia, and tendon) as well as well-differentiated chondrosarcomas had a similar staining reaction to that of adult cartilage (containing keratan sulphate). However, the intensity of the reaction was lower in these tumors than in the adult cartilage, indicating that the keratan sulphate content of the tumors is lower. Most of the moderately well-differentiated chondrosarcomas, the poorly differentiated chondrosarcomas, and pulmonary metastases of chondrosarcoma, as well as mesenchymal chondrosarcoma and extra-skeletal chondrosarcoma possessed the same staining properties as fetal cartilage, known to contain chondroitin 4- and 6-sulphate but not keratan sulphate. A few of the moderately well-differentiated chondrosarcomas stained up to a MgCl2 concentration of 1.0 M. Three cases of poorly differentiated chondrosarcomas stained with alcian blue up to 0.35-0.45 M in the lowest differentiated areas, indicating the presence of sulphated heteroglycans, as chondroitin 4- and 6-sulphate. Most chordomas possessed the same staining properties as fetal cartilage; however, a few chordomas stained in the same way as notochordal remnants of nucleus pulposus (containing keratan sulphate), which are thought to be the origin of these tumors. The results of staining of the tumors in the present series with the Scott technique corresponds well with toluidine blue and alcian blue at different pH's with and without pretreatment of the sections with testicular hyaluronidase. Since bone and soft tissue tumors may contain varying mucosubstances depending on the tissue of origin and on differentiation, histochemical investigation of the heteroglycan content of these tumors may be a valuable diagnostic aid.


The isolation of carcinoembryonic antigen from tumor tissue at neutral pH. Carcinoembryonic antigen (CEA) was purified from tumor tissue under mild conditions at neutral pH by a procedure that utilized affinity chromatography on concanvalin A. Further purification by gel filtration provided CEA in 10 to 20% yield and 10% purity. Antibody to this preparation was rendered specific for CEA by adsorption on a column of normal liver proteins bound to Sepharose. On reaction by immunodiffusion against a crude tumor extract, the adsorbed antibody produced two precipitin lines, of which one was relatively weak. These two precipitin lines fused completely with the two respective lines produced by antibody to perchloric acid-treated CEA. The major antigen found in crude tumor extracts and in CEA preparations purified at neutral pH was nearly undetectable in perchloric acid extracts of tumor homogenates. Further investigations showed that 60 to 70% of the CEA in crude tumor extracts and in preparations isolated at netural pH is destroyed and/or becomes insoluble acidic conditions.


Functional and structural alterations of liver ergastoplasmic membranes during DL-ethionine hepatocarcinogenesis. Different functional and structural properties of rat liver microsomes were studied during hepatocarcinogenesis induced by 0.25% DL-ethionine. During the first to fourth months of ethionine feeding, great decreases of cytochrome P-450 content, reduced nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation, and animopyrine demethylase activity occurred. No changes in the reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity were observed. These functional alterations were paralleled by an increase in membrane-free  ribosomes and by changes in the relative proportions of phospholipid fatty acids in microsomes. After the end of ethionine feeding, when hyperplastic nodules and/or hepatomas were present, the above functional and structural parameters were studied in the latter tissues, as well as in surrounding nonodular liver. Decreases in cytochrome P-450 content, lipid peroxidation, and animopyrine demethylase activity were documented in hyperplastic nodules and hepatomas. In hepatomas, the alterations were more marked and decrease of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was also found. All these functional parameters were quite normal in surrounding nonnodular liver. Similarly, alterations in phospholipid fatty acid composition disappeared in surrounding nonnodular liver, but they partially persisted in both hyperplastic nodules and hepatomas. In contrast, the increase in membrane-free ribosomes also occurred in surrounding nonnodular liver, although to a lower extent than in hyperplastic nodules and hepatomas. These data are discussed in relation to the problem of the cellular precursors of hepatomas.


Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). The behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14) was determined in normal, differentiating, and regenerating liver and in a spectrum of hepatomas of widely different growth rates. The liver and tumor enzymes were measured in 100,000 x g supernatants prepared from 20% tissue homogenates containing 0.25 M sucrose and 1 mM MgC12. Kinetic studies were carried out on the amidotransferase in the curde supernatant from liver and rapidly growing hepatoma 3924A so that under optimum standard assay conditions only the enzyme amount would be the limiting factor. The kinetic results showed that certain properties of the amidotransferase from liver and hepatoma were similar. The liver and hepatoma enzyme exhibited apparent Km's for: glutamine, 1.7 and 2.3 mM; MgC12, 0.7 and 1.1 mM, and phosphoribosylpyrophosphate. S0.5 for 0.9 and 0.4 mM, respectively...


Microsomal metabolism of triazenylimidazoles. The antitumor agents 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide (DIC) and 5-[3,3-bis(2-chloroethyl)-1-triazenyl]imidazole-4-carboxamide (BIC) are substrates for NADPH-requiring microsomal enzymes of mouse liver. The products of DIC oxidation are 5-aminoimidazole-4-carboxamide (AIC) and formaldehyde. Those for BIC are AIC and, presumably, 2-chloroacetaldehyde. For DIC, the reaction has a pH optimum of 9.0; and the Michaelis constant (Km) is 0.25 mM. At lower pH values, the Km is not greatly increased; but there is a sharp rise in the Km values above pH 9.0. For the enzyme-catalyzed production of AIC from BIC, the pH optimum is 7.5; the Km value for BIC is 0.47 mM. Of a variety of tissues tested for enzymatic activity, only liver accomplishes the conversion of DIC and BIC to AIC. Most of the activity in the liver is located in the microsomal fraction, although detectable activity is present in washed mitochondria. For liver microsomes, the rate of reaction for BIC is greater than that for DIC, but apparently neither rate is fast enough to allow extensive metabolism of large doses of these agents.


The effect of cupric acetate on ethionine metabolism. The addition of cupric acetate, a potent inhibitor of ethionine carcinogenesis, to a diet containing ethionine increased the ethionine toxicity. The concentration of S-adenosylethionine in liver was found to be significantly higher when compared to animals fed only ethionine in the diet. Ethionine forms a complex(es) with cupric acetate that is insoluble at a pH higher than 4; however, this complex can be solubilized at a low pH. Ethionine, if administered p.o. in the form of this complex, was absorbed from the intestinal lumen in the same order of magnitude as when administered alone; however, as the body weight increased over 200 g, the portion of absorbed ethionine decreased. The absorption of ethionine bound in the complex was completed within 16 hr compared to 2 hr for free ethionine. This time delay was accompanied by a shift in the concentration maximum of ethionine metabolities in the liver form 8 to 24 hr. When ethionine was administered alone, it was metabolized in the intestinal lumen as demonstrated by the analysis of the soluble intestinal contents; the presence of cupric acetate inhibited this process. The chromatographic analysis of ethionine metabolites in urine of rats treated by the complex revealed an increased excretion of ethionine sulfoxide and other ethionine metabolities at the expense of N-acetylethionine sulfoxide. The increased concentration of S-adenosylethionine in the liver in chronic experiments may be, at least partly, a result of a diminished capacity of the rat to detoxify (acetylate) ethionine sulfoxide, which is considered the main reserve pool of ethionine for the maintenance of a high level of S-adenosylethionine.


Effect of added nucleophiles and pH on alpha-D-mannosidase-catalyzed reactions. The effect of (1) added nucleophiles, (2) the concentration of water, and (3) pH on the alpha-D-mannosidase-catalyzed hydrolysis of substituted phenyl alpha-D-mannopyranosides has been investigated.. A two-step mechanism, with formation of a mannosyl-enzyme complex, is proposed. With the phenyl alpha-D-mannopyranosides, the formation of this intermediate complex is the rate-limiting step. The activity of the enzyme is controlled by two dissociable groups having pK approximately 3 and pK approximately 6, respectively. It is proposed that one of the groups functions as a proton-donor, whereas the other group stabilizes the mannosyl-enzyme complex by ion-pair formation or covalent binding.


Evidence for an increase in adrenergic nerve function in blood vessels from experimental hypertensive rabbits. The possibility of changes in the adrenergic innervation of blood vessels in experimental hypertension was investigated by measuring arterial norepinephrine content, neuronal uptake of norepinephrine, and the neurogenic contractile response in rabbits made hypertensive by partial constriction of the abdominal aorta proximal to the kidneys. Two to 3 weeks after surgery, norepinephrine content was increased in the arteries above the ligature, where arterial blood pressure was increased, but not in the arteries below the ligature, where arterial blood pressure was normal, in the heart, or in the veins. Neuronal norepinephrine uptake per unit length of vessel and the neurogenic contractile response increased with the rise in arterial blood pressure. The neurogenic contractile response can be taken as an indication of an increase in transmitter release. The results taken together suggest an increase in the function and possibly the amount of the adrenergic neuroneal terminal in hypertension. Since the distributions of the changes in the adrenergic innervation and the increases in smooth muscle cell proliferation in hypertension are similar, these two processes may be interrelated.


Colorimetric measurement of ornithine carbamoyl transferase activity in plasma, and results for a supposedly healthy population. Determination of ornithine carbamoyl transferase (EC 2.1.3.3) activity in plasma is important for detection of liver diseases. The assay established in this paper has been made optimum. A blank is needed containing both substrates, carbamoyl phosphate and ornithine. We used a new colorimetric assay, based on a complex with a phosphoferric-antipyrine reagent and diacetyl monoxime, to measure the citrulline formed. The highly sensitive assay permits low activities to be determined accurately. Values for blood plasma from 425 supposedly healthy people, varied from 0 to 16 U/liter (95th percentile), and 27% of this population showed an activity of less than 2 mumol of citrulline formed per minute per liter, 2 U/liter being the limit of the method's sensitivity.


Continuous determination of the oxygen dissociation curve for whole blood. We report here the development of a new method that allows continuous determination of the oxygen dissociation curve for microsamples (600 mul) of whole blood under conditions of pH, pCO2, methemoglobin concentration, and 2,3-diphosphoglycerate content closely approaching those found in the circulatory system. The method consists of gradually oxygenating a blood sample by adding H2O2 in the presence of catalase (EC 1.11.1.6), to produce the reaction H2O2 leads to H2O + 1/2 O2. Because the total oxygen content of blood can be derived from the known rate of H202 addition and the pO2 is determined in the liquid phase by an oxygen electrode, the two functions (total O2 content) and (% oxygen saturation) vs. pO2 are simple to calculate. pCO2 and pH are controlled by adding base simultaneously with the gradual oxygenation of blood. The method described thus avoids the direct measurement of oxygen saturation of whole blood.


Hepatic synthesis of canavaninosuccinate from ureidohomoserine and aspartate, and its conversion to guanidinosuccinate. This study continues the exploration of the mechanism for the formation of guanidinoacetate and guanidinosuccinate in the human [Clin. Chem. 21, 235 (1975)]. In this report we describe the formation of canavaninosuccinate from ureidohomoserine and aspartate by a human or bovine liver extract that had high argininosuccinate synthetase (EC 6.3.4.5) activity, and the subsequent formation of guanidinosuccinate by reductive cleavage. In the presence of ATP the optimum pH for the synthetic reaction is 8.4. This reaction can be carried out in either a tris(hydroxymethyl)aminomethane or borate buffer. Subsequent addition of dithiothreitol in the presence of Fe2+ resulted in the cleavage of some of the synthesized canavaninosuccinate to form guanidinosuccinate and homoserine. Synthesis of canavaninosuccinate was strongly inhibited by added argininosuccinate, less so by canavaninosuccinate, arginine, canavanine, glycine, or 2,3-dimercaptopropanol. The Km values for the substrates of the synthetic reaction are 3.6 X 10(-4) mol/liter for aspartate, 1.6 X 10(-3) mol/liter for ureidohomoserine, and 2.92 X 10(-5) mol/liter for ATP. These values are higher than those obtained when the synthesis of argininosuccinate was studied, except for ATP, which yielded a lower value. All of the reactions in the proposed mechanism have now been demonstrated except for the synthesis of canaline from aspartate.


Biochemical activity in the metastatic lesions of neuroblastoma. Catecholamine biosynthesis in tumor cells was studied in four patients, who expired from disseminated neuroblastoma. 1. The activity of tyrosine hydroxylase was detected in tumor tissues of all but one patient. 2. Dopamine-beta-hydroxylase activity was assayabel in almost all tissues, except in the primary tumor of one patient. 3. The enzyme activities in the metastatic lesions were at the same levels in two cases, but much higher in the other two cases, as those in the primary tumours. 4. It was learned that the urinary excretion of vanilmandelic acid (VMA) and homovanillic acid (HVA) in these patients reflects the tissue levels of catecholamine synthesizing enzymes as well as the tumor mass.


Fabry disease: diagnosis by alpha-galactosidase activities in tears. The enzymatic diagnosis of hemizygotes with Fabry disease and heterozygous carriers was accomplished by the fluorometric determination of alpha-galactosidase activities in tears. Two components of total alpha-galactosidase activity were differentiated by their relative thermostabilities and by chromatography on DEAE-cellulose. The major component, alpha-galactosidase A, was thermolabile and represented approximately 90% of total activity; the remaining activity was thermostable, eluted at a slightly higher salt concentration and was designated alpha-galactosidase B. A single, symmetric pH optimum was observed for total alpha-galactosidase activities from heterozygotes and normal individuals, whereas the total activity from hemizgotes, which was about 10% of that in normal controls, had a broad pH profile, identical to those for alpha-galactosidase B activities from all individuals studied. The apparent Km values for total activities were 3.2, 4.0, and greater than 13 mM for normal individuals, heterozygotes, and hemizygotes, respectively. In contrast, apparent Km values for alpha-galactosidase B activities were greater than 13 mM for all individuals, further suggesteng that the residual activity in hemizygotes with Fabry disease represented the alpha-galactosidase B component. of the potential inhibitors studied, alpha-D-melibiose was found to competitively inhibit total alpha-galactosidase activity (Ki approximately 10 mM). These studies demonstrate that tears provide an easily obtainable source of freshly secreted enzyme for the diagnosis of hemizygotes and heterozygotes with Fabry disease and suggest that tears may be useful for the diagnosis of other inborn errors of metabolism.


An alternative method for the determination of uric acid in serum. A novel fluorometric method for the determination of uric acid based on the coupled reactions of uric acid with uricase and peroxidase to form highly fluorescent 2,2'dihydroxy-3,3'-dimethoxy-biphenyl-5,5'-diacetic acid is described. The calibration curve was constructed from a series of standard uric acid solutions vs. the corresponding relative fluorescence. It was linear up to 15 mg/dl (0.9 mmol/l). The serum or plasma samples must be deproteinated with (absolute) ethanol before assay and its uric acid content can be obtained from the calibration curve. This method is rather simple, having good precision and accuracy. High level ascorbic acid in the sample falsely elevates uric acid concentration. Comparison of the results obtained on the patient sera with a colorimetric phosphotungstate method and a standard enzymatic spectrophotometric method gave coefficients of correlation of 0.908 and 0.986, respectively.


Serum ionized calcium by electrodes: new technology and methodology. We report experience measuring serum ionized calcium (Ca2+) with new solid-state, specific calcium ion dip electrodes in a commercially available system for automated electrode measurments which permits stable measurements on thirty samples with up to six different electrodes simultaneously. The system provides electrostatic shielding, temperature regulation at 37degreeC, and adjustment of serum pH with CO2 gas, thereby making practical the use of serum standards and routine measurements on sera. When the electrodes are used with the rest of the measuring system and with serum standards, the system provides serum Ca2+ measurements with high precision (C.V. of 0.7%) and the calcium electrodes maintain good slopes for 50 to 150 hours of cumulative wet immersion. Response to new calcium activity is rapid, and wide variations in serum Ca2+ are readily measured.


A new variant of glucosephosphate isomerase deficiency (GPI-Utrecht). A new case of glucosephosphate isomerase deficiency is described in a Dutch family. The activity of the enzyme was decreased to 20-25% of the normal value. Characterization of the defect enzyme showed a pronounced thermolability. Heating of the enzyme at 45 degrees C showed a loss of activity of 90% after one hour. The pH-optimum and the electrophoretic migration were normal. The Km-value for F-6-P, the Ki for the competitive inhibitors 2,3-DPG and 6-PG were in the normal range. The variant described here differs from all known variants. Therefore we propose to give to this new variant the name of GPI-Utrecht.


Studies on the stability of pancreatic enzymes in duodenal fluid to storage temperature and pH. The effects of storage pH and temperature on pancreatic amylase, lipase and trypsin in duodenal fluid were studied. All the enzymes were most stable when stored at --20 degrees C, while the optimum storage pH was found to depend on the particular enzyme under investigation.


Zonal ultracentrifugation of plasma lipoproteins from normal and cholestatic pigs. The plasma lipoproteins in normal and cholestatic pigs were isolated by zonal ultracentrifugation and studied with respect to apoprotein and lipid composition. In contrast with the distribution in human plasma, only one HDL-population but two LDL-populations were demonstrated. No cholestatic lipoprotein similar to the human lipoprotein X was observed. HDL and the lighter LDL component were largely unchanged in cholestasis. The immunochemical properties were changed to some extent. Thus HDL-reacting material was found in the heavier LDL-component and VLDL after cholestasis but not before.


[A kinetic method for the determination of the activity of "aerobic transhydrogenases" (author's transl)]. The chromogen ABTS is the di-ammonium salt of 2,2'-azino-di[3-ethyl-benzthiazolin-sulfonic acid (6)] routinely used in the "glucose-oxidase assay" with the peroxidase (GOD-Perid method, Boehringer). 1. The specific property of ABTS to give a stable radical cation by oxidation with hydrogen peroxide in the presence of peroxidase was used to design a kinetic method, for enzyme-activity determinations. 2. The assay is suitable for the specific oxido-reductase using oxygen as acceptor, known also as "aerobic transhydrogenases" which are H2O2 formers (EC 1.-.3.-). 3. L-Amino acid: oxygen oxidoreductase (deaminating) (EC 1.4.3.2), was used throughout, being a representative model for such determinations.


IgG and IgA antigens in human renal basement membranes. Cryostat sections of fresh human kidney studied by immunofluorescence were negative for IgG, IgA, IgM, Fc and Fab fragments of IgG, and kappa and lambda light chains. All of these conjugates except anti-IgM stained glomerular basement membranes and several other connective tissues if the cryostat sections were washed in phosphate-buffered saline (PBS) before being studied. Staining was inhibited by blocking reactions as well as by fixation before washing in PBS or by washing in non-ionized or hypotonic solutions. Results of several other aspects of this reaction are also reported.


Acetylation polymorphism of sulfapyridine in patients with ulcerative colitis and Crohn's disease. Sulfapyridine (SP) is one of the main metabolites of salicylazosulfapyridine (sulfasalazine) that is used extensively in the management of inflammatory bowel disease. One hundred and twenty-two patients with ulcerative colitis or Crohn's disease were studied, including 21 new, untreated patients and 101 previously treated patients. Patients were studied for at least one year during active disease and remission. It was shown that sulfapyridine shares the same acetylation polymorphism as sulfadimidine. The acetylation capability of each patient as determined in serum and urine was constant irrespective of dose (2 to 8 gm/day) and state of disease. A single study of serum can determine acetylator phenotype in patients on sulfasalazine therapy without using any other drug for this purpose and may help ascertain dosage and assess side effects.


Salivary excretion of amobarbital in man. The concentrations of amobarbital in saliva and serum were determined in 5 normal adults following ingestion of 120 mg of sodium amobarbital. There was excellent linear relationship between amobarbital concentrations in saliva and serum (r = 0.993); salivary levels were 36.1% of serum levels. Since the pH of saliva was generally lower than that of blood in man, the degree of ionization of amobarbital in serum and saliva had to be taken into consideration. Estimation of the protein binding of amobarbital in serum from concentrations of amobarbital in saliva and serum was in good agreement with the in vitro data of equilibrium dialysis.


The relationship of plasma guanethidine levels to adrenergic blockade. Seventeen hypertensive patients receiving guanethidine for therapy were studied to determine the relationship of guanethidine plasma levels to adrenergic blockade. Plasma levels of guanethidine were measured by gas chromatography-mass spectrometry, and adrenergic blockade was defined by determining the venous reflex response to Valsalva maneuver or deep breath. A significant correlation was found between the change in the venous reflex response and the fall in mean standing pressure when guanethidine is given to patients maintained on a sodium restricted diet. A linear relationship was found between dose and plasma guanethidine concentration (p less than 0.0001), but there was a 6-fold interindividual variation in the plasma levels resulting from any given dose. Adrenergic blockade occurred when plasma levels were 8 ng/ml or higher. These results indicate that the large individual variation in dose requirements for the hypotensive effects of guanethidine most likely is not due to requirements for greatly different plasma levels of the drug; that the variation must result from pharmacokinetic determinants of differing plasma levels between individuals or from other factors, such as increased plasma volume, which maintain elevated arterial pressure in the face of adrenergic blockade.


Effect of acidosis on patients with myocardial ischemia. Reduction in pH is known to decrease O2 affinity (Bohr effect) and increase the rate of O2 release from blood. It provides a potential mechanism for increasing O2 transport to the myocardium. Fifteen patients with refractory, chronic angina were studied by treadmill exercise tolerance tests and whole blood-oxygen release rate measurements before and 4 days after beginning treatment with oral acetazolamide (10 to 20 mg/kg body weight). Positive treadmill exercise test or myocardial necrosis was present in each case. There was a correlation between an increased O2 release rate from blood and relief of symptoms. The major side effect in 2 patients with pathologic fatigue believed to result from acidosis. Of the 7 patients who obtained relief from angina, in each there was a 27% increase in the rate of release of O2 from their whole blood. Seven patients did not obtain relief; they showed no change in the rate of release. In the 4 patients who became worse the rate of deoxygenation decreased by 22%. All changes in deoxygenation rate coincided with the clinical findings. The treatment of ischemic heart disease with acetazolamide or other acidifying agents should not, however, be attempted until further investigation establishes their clinical value.


Intracellular pH: measurement, control, and metabolic interrelationships. Recent work in the field of cell pH has been characterized by many developments in techniques of measurement, by increasing knowledge of the mechanism of control of cell pH, and by progress in the establishment of relationships between cell pHi and certain areas of intermediary metabolism. Though the weight of evidence is much in favor of control cell pH by active transport of H+, the situation remains somewhat unsatisfactory due to lack of a completely adequate explanation of the work of Carter's group. The heterogeneity of cell pH raises problems in the intrepretation of hydrogen ion equilibria across cell membranes and serious difficulties in correlating changes with alterations in metabolism. To put this into perspective, however, the later difficulties are no greater than many experienced with cell constituents other than hydrogen ions.90 As in other fields, knowledge must advance by making the best of the currently available methods until such time as better techniques become available.


The chemical and biological background to alclofenac. The essential chemistry of alclofenac is described together with a resumé of the pharmacological and toxicological properties. Its metabolism is described and absorption and excretion commented upon. The unique character of the alclofenac molecule in relation to its active allyloxy group and its close conformational resemblance to L-tryptophan is emphasised. Particular note is made of the absence of gastro-intestinal irritation in the long-term studies in animals and in man.


Influence of pharmacologic agents on tissue metabolism in circulatory shock. Our research activities have been oriented to the effect of pharmacologic agents on tissue metabolism in the low flow state of circulatory shock. Specifically, we have been most interested in the effect of glucocorticoids on cellular metabolism because these agents exert a reproducible protective effect during septic and endotoxic shock in rats and monkeys. Further, we have studied the differences between untreated animals in shock and those treated with pharmacologic agents in an attempt to determine which cellular metabolic changes are critical to survival. This paper will present our studies as well as those of other laboratories, which have defined the effects on tissue metabolism of certain pharmacologic agents: those which stimulate cyclic adenosine monophosphate (AMP) (epinephrine, norepinephrine, glucagon, and prostaglandin) and those which do not (the glucocorticoids).


A review of possible mechanisms of action of the antirheumatic drug, alclofenac. A number and variety of hypotheses have been proposed to explain the primary mechanism of action of the antirheumatic drugs. A critical review is made of the biochemical and pharmacological parameters which, on current evidence, parallel the clinical activity of the drugs. The numerous chemical mediators of the inflammatory response are discussed, as is the possible role of endoenous anti-inflammatory substances. Particular attention is paid to the plasma protein-binding effects of antirheumatic drugs and the competitive displacement theory of drug action is examined in the light of recent work on the behaviour of L-tryptophan binding in vitro and in vivo. It is suggested that antirheumatic drugs can be differentiated by their influence on the acute-phase protein response and their effects on tryptophan binding. Clinical findings are reviewed to support the proposal that drugs such as alcofenac, which both inhibit the acute-phase response and exert prolonged influence on the binding of tryptophan to plasma proteins, reduce inflammatory activity more profoundly than those drugs which produce symptomatic relief alone. They are capable, therefore, of altering the course of the disease.


Simultaneous pharmacokinetics of alclofenace in plasma and synovial fluid in patients with rheumatoid arthritis. The simultaneous pharmacokinetics of alclofenac in the plasma and synovial fluid of 10 patients with rheumatoid arthritis were studied after a single 1 g. oral dose. A gas-liquid chromatographic method was used for assaying alclofenac in both plasma and synovial fluid. Alclofenac readily appears in the plasma and synovial fluid.


Serum sulphydryl levels in rheumatoid patients treated with alclofenac. Serum sulphydryl levels, which are decreased in rheumatoid patients, are increased to near normal during treatment with alclofenac. The biochemical function of the sulphydryl group and its possible pathogenic significance in rheumatoid and other auto-immune diseases are discussed.


Clinical studies on alclofenac in the treatment of rheumatic diseases: a drug in question. The potential advantage to patients with chronic rheumatic diseases of an effective, non-steroidal analgesic/anti-inflammatory drug which causes insignificant gastric bleeding was a decisive factor leading to the introduction of alclofenac. Short-term double-blind trials showed that alclofenac has analgesic/anti-inflammatory activities equivalent to phenylbutazone, indomethacin and aspirin, but superior to the fenemates and propionic acid derivatives. Long-term controlled studies, ranging from 5 months to 3-1/2 years and using reliable, objective measures revela, however, that patients with rheumatoid arthritis improve in functional status and graduate to less severe classes of disease activity, a phenomenon not observed with either indomethacin or aspirin administered to matched patients over the same periods of time. So far, clinical improvement on alclofenac has been matched only by treatment with gold, D-penicillamine and the immunosuppressive anti-proliferative drugs. This clinical improvement on alclofenac is reflected in haematological and serological indices, and research shows that alclofenac, like these other antirheumatoid drugs, has a pronounced effect upon the acute-phase protein response and the extent to which L-tryptophan is bound to plasma protein. The clinical data reviewed suggest that alclofenac represents an advance in the therapy of the rheumatic diseases.


The influence of alclofenac treatment on acute-phase proteins, plasma tryptophan, and erythrocyte sedimentation rate in patients with rheumatoid arthritis. Alclofenac and D-penicillamine were compared under controlled, double-blind conditions in the treatment of 35 patients with active rheumatoid arthritis over a period of 26 weeks. The principal aim of the study was to investigate any relationships between changes in clinical status and changes in concentrations of three serum acute-phase proteins (fibrinogen, C-reactive protein, and haptoglobin), plasma free and protein-bound L-tryptophan, and the erythrocyte sedimentation rate. Both alclofenac and D-penicillamine were clearly effective: all patients showed steady improvement on the seven clinical indices of response employed. Drug management was easiest with alclofenac. Both drugs produced a significant reduction in acute-phase proteins, E.S.R. and protein-bound plasma tryptophan. Since it has previously been established that the course of rheumatoid arthritis is reflected in the acute-phase protein levels and the extent to which L-tryptophan is bound to plasma protein, it is suggested that drugs, such as D-penicillamin and alclofenac which profoundly affect these parameters, provide not only symptomatic relief but also possible beneficial effects on the disease process itself.


The effect of alclofenac on clinical and laboratory measures of disease activity in rheumatoid arthritis: preliminary results. The results are presented for the first 11 patients in an on-going 3-month study of 1 g. alclofenac t.d.s. or matching placebo added to the drug regime of patients with active rheumatoid arthritis uncontrolled by full doses of analgesic/anti-inflammatory medication. Two patients dropped out, 1 due to a rash on alclofenac and 1 for incidental reasons. The numbers at this stage are too small for meaningful statistical comparison, but show an apparent trend towards clinical improvement and increase in the free:bound plasma tryptophan ratio in the alclofenac-treated patients. Three acute phase proteins, C-reactive protein, alpha-acid glycoprotein and alpha-1 antitrypsin, were measured. They did not correlate with the E.S.R. and did not appear to be useful additional predictors of disease progression. It is hoped that the results from the rest of the participants in the trial will allow more meaningful discussion of the place of these tests in the assessment of rheumatoid arthritis.