Ectopic apudocarcinomas and associated endocrine hyperplasias of the foregut.

Foregut endocrine polypeptide-secreting APUD cells (Amine-Precursor-Uptake and Decarboxylation), in their embryologic migration from neural crest to foregut may become "arrested" in the mesoderm or in other ectopic locations. They may become hyperplastic, adenomatous or malignant. Eight illustrative patients are reported. One patient had "pancreatic hyperparathyroidism" with hypercalcemic crises, pancreatic apudocarcinoma, normal parathyroids, biologically active parathormone, but inert immunochemically to the usual parathyroid antisera. Two had gastrin-secreting malignancies in the mesoderm. Remission after excision, but eventual recurrence of the syndrome due to islet cell hyperplasia required total gastrectomy. One patient had a gastric corpus apudocarcinoma found prospectively with hypergastrinemia which required excision of the tumor. One patient had acromegaly with hypergastrinemia and antral gastrinosis treated by pituitary irradiation,  One patient had the antral or intermediary type of the Zollinger-Ellison syndrome with moderate hypergastrinemia, duodenal ulcer and antral gastrinosis, treated by vagotomy and antrectomy. One patient had hyperparathyroidism with antral gastrinosis, treated by parathyroidectomy. One patient had malignant Zollinger-Ellison syndrome and developed associated thyroid parafollicular cell hyperplasia and parathyroid chief cell hyperplasia, treated by total gastrectomy and multiple endocrine excisions. These investigative observations demonstrate ectopic loci and associated hyperplasias which support the concept of migration and bizarre potentiality of polypeptide-secreting cells of the foregut.


A study of the effect of d-amphetamine on the toxicity, analgesic potency and swimming impairment caused by potent analgesics in mice.

The addition of d-amphetamine to morphine has been reported to result in an increase of analgesic potency in experimental animals and man, but no data on the toxicity of such combinations are available. This study is intended to provide systematic information on the toxicity, analgesic potency and degree of physical impairment (swimming endurance) of a combination of 12 mg morphine sulfate with 10 mg d-amphetamine HCl per ml which is now under clinical investigation. Mice were used as experimental subjects. Meperidine, methadone and pentazocine were substituted for morphine using clinically equally analgesic doses and keeping the d-amphetamine amount constant. The toxicity of all analgesics especially that of morphine was enhanced in the combination, least so in the case of meperidine. The degree of increase of analgesic power by the addition of d-amphetamine was greatest with morphine and quantitatively in satisfactory agreement with present clinical experiences. However, the relationship between the increases of toxicity and of analgesia is not necessarily most favorable for this drug. For the other three analgesics increases in toxicity and analgesia were more in line, meperidine showing the best ratio. Swimming endurance was decreased with full analgesic doses of all four compounds. The presence of d-amphetamine tended to reverse this depression. The data were analyzed in relation to their possible predictive value for the use of such combinations in man for the therapeutic dose range and in the event of overdosage.


[The calorigenic effect of glucagon].

The calorigenic action of glucagon was studied in several species by measuring its effect on the oxygen comsumption. The calorigenic effect was most pronounced in the quail and also evident in young adult rats of two different strains but not in mice and guinea pigs. The influence of various drugs on the calorigenic effect of glucagon was investigated in rats. Ganglionic blocking agents (hexamethonium, mecamylamine, chlorisondamine) as well as adrenergic blocking drugs (propranolol, butoxamine, phentolamine) greatly reduced the glucagon-induced calorigenesis but after pretreatment with reserpine, guanethidine or chemical sympathectomy with 6-hydroxydopamine the calorigenic effect of glucagon was exaggerated. It was somewhat diminished by nicotinic acid but not by chlorpromazine or lithium and potentiated by cocaine but not by theophylline.--These results are not indicative for an involvement of adenly cyclase--cyclic AMP--system in glucagon-induced calorigenesis but they are compatible with the assumption that the hormone enhances the metabolic rate by releasing catecholamines from the adrenal medulla.


Anxiety in schizophrenia. The responses to chlordiazepoxide in an intensive design study.

Six anxious schizophrenic patients who were maintained with phenothiazines participated in a double-blind intensive design study of chlordiazepoxide and placebo for 12 weeks or longer. There were substantial differences between patients in their responses to chlordiazepoxide: two patients experienced significant and conspicuous relief of distress and reduction of typical schizophrenic symptoms. In another patient the differences, although statistically significant, were clinically less striking. In the three remaining patients no differences were observed between responses to the two treatments except that one patient was more depressed with chlordiazepoxide than with placebo. The findings suggest that there are at least two kinds of anxiety in schizophrenia. A few anxious schizophrenic patients apparently benefit more from a combination of a phenothiazine with chlordiazepoxide than from a phenothiazine alone.


Behavioral Effects of Reducing the Daily Frequency of Phenothiazine Administration.

After an 11-day base line of behavioral observations, 24 chronic female schizophrenics were assigned to two groups matched for alertness. In the first treatment phase, the administration of phenothiazine medication of one group was switched from a multiple-dose schedule (three to four times per day) to a single daily administration, while the total dosage per day was held constant. The second group continued on a multiple administration schedule for 11 days and then was switched to a single daily dosage. A multivariate analysis of variance showed that there was no overall effect (positive or negative) due to the schedule change; however, preplanned t tests showed transitory decreases in nonfunctional behavior. The results are discussed in terms of implications for the administration of phenothiazines and the experimental analysis of drug effects.


The basis for amine hypotheses in affective disorders. A critical evaluation.

A leading hypothesis concerning a biological basis of the affective disorders is that altered metabolism of brain amines may underlie the cause or pathophysiology of these conditions. Features of affective illnesses supporting biological hypotheses include the somatic symptoms, diurnal rhythm, and apparent "endogenicity" of many severe depressions, and evidence of a genetic basis of manic-depressive illness. Development and preclinical study of medical therapies for the disorders substantially supported a relationship between mood-disturbances and neutrotransmitters and stimulated considerable advances in the physiology and pharmacology of central synaptic neurotransmission. Unfortunately, studies of amine metabolism in patients have not provided consistent support for the amine hypotheses. Moreover, these hypotheses have not led to a coherent biological theory of abnormal behavior, to an objective basis for differential diagnosis, or to the rational development of treatments more effective or safer than those known.


[Benign neurologic accident following anti-rabies vaccination. Unforeseen clinical and biological aspects].

A minor and benign encephalitogenic reactions was observed in the days following administration of suckling mouse brain anti-rabies vaccine. The syndrome appeared at the 18th day of the treatment, undertaken according to Gamet and Atanasiu's shortened treatment schedule. Recovery occured without sequellae after 5 days of corticosteroid therapy. Compared to the reports of the literature, the benignity of the central nervous system involvement was unexpected. Three immunological problems are stressed out: the presence of sheep anti-red blood cells antibodies after antirabies vaccines; the presence of antismooth muscle antibodies; a previous BCG vaccination one month before antirabies treatment might play an adjuvant participation.


Antigenic comparison of swine influenza virus isolates.

Cross hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests demonstrated strong antigenic relationship between A/Swine/Wisconsin/1/73 (SW/73) and A/Swine/Shope/15/31 (SW/31) influenza viruses. An eigthyone fold purification of virus was achieved by adsorption and elution followed by differential ultracentrifugation and sedimentation through linear sucrose gradient. Radioimmunoassay using purified 125I labeled viral antigens revealed antigenic variation between the two virus isolates. Neuraminidase of both viruses had pH optima between 6.5 and 7.0, and SW/31 enzyme was relatively more heat stable than SW/73.


The isolation of aggregates of spectrin from bovine erythrocyte membranes.

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4% agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960 000 and 480 000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physical properties of spectrin.


Pyridine nucleosidase in bull semen. II. Biochemical properties.

It is most likely a single enzyme (NAD+ nucleosidase) present in semen from most bulls which hydrolyses the ribosyl pyridinium bond in both NAD and NADP. This conclusion is based on the following results: (i) each of 12 semen samples containing nucleosidase activity hydrolysed NAD at the same rate as NADP (r = 0.99); (ii) other untreated semen samples from different bulls which did not hydrolyse NAD were also inactive against NADP; (iii) enzyme denaturation produced by preliminary heating of semen filtrates for 15 min at varied temperatures or by heating at 55 degrees C for varied time intervals caused similar reductions in the rates of NAD and NADP hydrolysis; and (iv) nicotinamide inhibited enzyme activity to the same degree using either NAD or NADP as the substrate.


Analysis of gases and pH of blood at altitude.

The accuracy of a system for measuring pH and gas in blood in a changing-altitude environment was studied. No problems were encountered when arterial blood was measured, provided the instrument was properly calibrated. Failure to calibrate to the correct altitude settings caused major variations in measuring oxygen levels in blood. Standard gases used for calibrating were measured at altitude without recalibrating the system; approximately 80% of the oxygen and 90% of the carbon dioxide partial-pressure values were within 3 mm Hg of their expected value. More deviation occurred for Po2 values as the altitude increased. Frequently, the Po2 electrode malfunctioned, particularly above 8000 ft (2438 m). The Corning Blood Gas System, which was used in this study, was capable of making accurate determinations of gas in blood if properly operated and calibrated to specific altitude levels.


Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.


The preparation and some properties of mammalian cytochrome c modified with 2-hydroxy-5-nitrobenzyl bromide.

2-Hydroxy-5-nitrobenzyl bromide reacts with horse heart cytochrome c at acid pH to yield a chemically modified protein. Chromatography of the protein on CM-cellulose allows separation of a single chemically modified species. This species is shown by gel chromatography to be monomeric, and isoelectric focusing shows the pI to be lowered from 10.5 to 9.8 on introduction of the reagent molecule. The changes observed in the u.v. region of the spectrum are consistent with the introduction of a single residue of the reagent, and the normal fluorescence of tryptophan is lost. The chemically modified protein exhibits marked changes in its functional properties as compared with native cytochrome c. Unlike the native monomer, the modified cytochrome c has a pH-dependent spectrum which is typical of a high-spin species in the alpha/beta region at low pH, changing to a low-spin species with an apparent pK of 7.5. The modified protein is autoxidizable and the ferrous form binds CO at neutral pH with an affinity constant of 2.6 X 10(5)M-1. The ferrous form of the modified cytochrome c binds CN- at pH 10.0 with an affinity constant of 3.5 X 10(2)M-1. The modified cytochrome c was incapable of restoring the electron-transfer activity to mitochondria depleted of cytochrome c.


Ethylenediamine-NN'-tetra-acetate-dependent amino acid-stimulated inactivation of mouse ovarian alkaline phosphatase activity.

1. Preincubation of partially purified preparations of mouse ovarian alkaline phosphatase in the presence of both EDTA and glycine at alkaline pH resulted in a pronounced inactivation of alkaline phosphatase activity. Inactivation did not occur on preincubation with EDTA or glycine alone. 2. The rate of inactivation was first-order with respect to the concentration of enzyme, and was independent of EDTA concentration above a threshold value. 3. The process was pH-dependent with a pK at 9.85, and inactivation was not dependent on the stereochemistry of the amino acid. A free alpha-amino group and a free carboxyl group at a specific spatial separation were essential for inactivation. 4. Inactivation involved the formation of an enzyme--metal ion--amino acid complex, the amount formed being dependent on both the nature and concentration of the amino acid. This complex then decayed to a derivative that was then acted on by EDTA, yielding an inactive form of the enzyme.


Acetylation of S-substituted cysteines by a rat liver and kidney microsomal N-acetyltransferase.

1. An acetyl-CoA--S-substituted cysteine N-acetyltransferase in rat liver and kidney preparations was investigated, by using an assay involving incubations with S-benzyl-L-cysteine and [l-14C]acetyl-CoA and extraction of the radioactive product with ethyl acetate. 2. The enzyme was associated with the microsomal fraction and could not be solubilized. Metal ions, EDTA and detergents did not significantly affect the enzyme activity. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme. 3. Other S-substituted cysteines were acetylated at about the same rate as S-benzyl-L-cysteine. Acetylation of cysteine itself and of methionine, ethionine and tryptophan could be detected but was much slower. Acetylation of aspartic acid, glycine, phenylalanine and serine could not be detected. Palmitoyl-CoA was not a substrate. 4. The enzyme is presumably responsible for the acetylation step of mercapturic acid synthesis; a more physiological function is not yet known, except that the enzyme may be involved in acetylation of those amino acids which occur in elevated amounts in some disorders of amino acid metabolism.


A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates.

The kinetics of ethanol oxidation by NAD+, and acetaldehyde and butyraldehyde reduction by NADH, catalysed by yeast alcohol dehydrogenase, were studied in the pH range 4.9--9.9 at 25 degrees C and in the temperature range 14.8--43.5 degrees C at pH 7.05. The kinetics of reduction of acetaldehyde by [4A-2H]NADH at pH 7.05 and pH 8.9 at 25 degrees C were also studied. The results of the kinetic experiments indicate that the mechanism of catalysis, previously proposed on the basis of studies at pH 7.05 and 25 degrees C (Dickinson & Monger, 1973), applies over the wide range of conditions now tested. Values of some of the initial-rate parameters obtained were used to deduce information about the pH- and temperature-dependence of the specific rates of combination of enzyme and coenzymes and of the dissociation of the enzyme--coenzyme compounds. Primary and secondary plots of initial-rate data are deposited as Supplementary Publication SUP 50043 (20 pages) with the British Library (Lending Division), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies may be obtained under the terms indicated in Biochem. J. (1975) 145, 5.


The purification and properties of L-histidine--2-oxoglutarate aminotransferase from Pseudomonas testosteroni.

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5'-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium  imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.


Ligand binding to ferrocytochrome c at high pH.

Ferrocytochrome c has been shown to bind two molecules of CO at pH 14. The second CO is thought to be bound only when the cytochrome c molecule is denatured, and once bound appears to be spectrally silent. Insolubilization of native cytochrome c prevents the binding of the second CO molecule. A scheme is proposed to explain these observations based on evidence from static titrations and flash-photolysis experiments, use of carboxymethyl cytochrome c and insoluble cytochrome c, and use of cyanide instead of CO as a ligand.


A study of the kinetics of iron and copper binding to hen ovotransferrin.

The kinetics of iron and copper binding to hen's-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.


Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism.

The use of a linear free-energy relationship shows that both histidine residues of alpha-chymotrypsin and chymotrypsinogen are super-reactive toward 1-fluoro-2,4-dinitrobenzene. The binding of indole to the specificity site of alpha-chymotrypsin causes both histidine residues to become less reactive. On the basis of these results and those from X-ray-crystallographic studies, the following conclusions are made. (1) The super-reactivity of the catalytic-site histidine-57 is due to charge transfer from aspartic acid-102 by means of hydrogen bonding. (2) The aspartic acid-102-histidine-57-serine-195 'charge-relay' system is not complete in the zymogen or native enzyme and only on binding of a suitable substrate or ligand to the specificity site of the enzyme is the charge transfer to serine-195 completed. (3) The lack of substantial enzymic activity in the zymogen is due to the absence of a completed specificity site, and therefore it cannot bind suitable substrates or ligands to induce completion of the charge-relay system.


Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.

A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.


Purification and properties of S-alkyl-L-cysteine lyase from seedlings of Acacia farnesiana Willd.

1. An S-alkyl-L-cysteine lyase (EC 4.4.1.6) was purified to apparent homogeneity from extracts of acetone-dried powders of the hypocotyls of etiolated 5-day-old seedlings of Acacia farnesiana Willd. 2. The enzyme catalyses a beta-elimination reaction and will utilize both the thioether and sulphoxide form of the substrate. 3. There is a braod specificity with regard to the alkyl substituent, but cystathionine is utilized very poorly. 4. The pH optimum is 7.8 and the Km value for the probable natural substrate L-djenkolate is 0.3 mM. 5. Both sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ultracentirfugal analysis give a molecular weight of about 144000. 6. One mol of pyridoxal phosphate is bound/mol of enzyme. 7. The energy of activation with L-djenkolate as the substrate is 53.1 kJ/mol. 8. The enzyme has a partial specific volume of 0.56 and S20,w 7.26S.


Selective inhibition of cholesterol synthesis by cell-free preparations of rat liver by using inhibitors of cytoplasmic acetoacetyl-coenzyme A thiolase.

Cytoplasmic acetoacetyl-CoA thiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) was partially purified from rat liver. The enzyme was irreversibly inactivated by 4-bromocrotonyl-CoA, but-3-ynoyl-CoA, pent-3-ynoyl-CoA and dec-3-ynoyl-CoA. In the case of the alk-3-ynoyl-CoA esters the potency as alkylating agents of acetoacetyl-CoA thiolase decreased with increased chain length of the alk-3-ynoyl moiety. Advantage was taken of the specific action of alk-3-ynoyl-CoA esters on acetoacetyl-CoA thiolase to show that in a postmitochondrial fraction from rat liver they are effective inhibitors of cholesterol synthesis from sodium [2-14C]acetate under conditions when mevalonate conversion into cholesterol and fatty acid synthesis are unafffected. Short-chain alk-3-ynoic acids have little effect on sterol synthesis, although dec-3-ynoic acid is an effective inhibitor owing to its conversion into the CoA ester by the microsomal fatty acyl-CoA synthetase.


pH-dependence of 13C chemical shifts and 13C,H coupling constants in imidazole and L-histidine.

The pH-dependence of selected 13C chemical shifts reflects the state of ionization of the imidazole ring in both imidazole and L-histidine. Titration of the amino and carboxyl groups of histidine also perturbs the shifts. The coupling constants 1J (13C(2),H) and 1J (13C(5),H) for both compounds also vary with pH, but in L-histidine these constants are relatively insensitive to the titration of groups outside the imidazole ring.


Hydrodynamic changes accompanying the loss of metal ions from concanavalin A.

The hydrodynamic changes which accompany the dissociation of metal ions, from concanavalin A at acid pH are a result of charge effects rather than of dissociation of metal ions as such. Measurements of the rotational relaxation time are discussed in terms of the hydration of the protein and its polymeric heterogeneity.


Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.


Unidirectional inhibition and activation of "malic' enzyme of Solanum tuberosum by meso-tartrate.

A kinetic study of "malic' enzyme (EC 1.1.1.40) from potato suggests that the mechanism is Ordered Bi Ter with NADP+ binding before malate, and NADPH binding before pyruvate and HCO3-. The analysis is complicated by the non-linearity that occurs in some of the plots. meso-Tartrate is shown to inhibit the oxidative decarboxylation of malate but to activate the reductive carboxylation of pyruvate. To explain these unidirectional effects it is suggested that the control site of "malic' enzyme binds organic acids (including meso-tartrate) which activate the enzyme. meso-Tartrate, however, competes with malate for the active site and thus inhibits the oxidative decarboxylation of malate. Because meso-tartrate does not compete effectively with pyruvate for enzyme-NADPH, its binding at the control site leads to a stimulation of the carboxylation of pyruvate. A similar explanation is advanced for the observation that malic acid stimulates its own synthesis.


Affinity chromatography of immobilized actin and myosin.

Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.


Effect of environmental stress of low pressure on tyrosine aminotransferase and phenylalanine 4-hydroxylase activities in the rat.

1. Tyrosine aminotransferase activity in the liver increased about fourfold after 9h, on exposure of rats to stress of low pressure. 2. The phenylalanine hydroxylase activity increased about 60% on exposure for 24h or more. 3. An environmental pressure decrease of about 0.033 MN/m2 is needed to increase the activity of tyrosine aminotransferase. 4. Adrenalectomy completely abolished the increase in activity of tyrosine aminotransferase obtained on exposure to low pressure. 5. Treatment with cycloheximide or actinomycin D prevented the increase in activity of tyrosine aminotransferase. 6. Treatment with cycloheximide at the early part of exposure to stress prevented the increase in activity of phenylalanine hydroxylase obtained after 24h.


A study of the effects of propranolol and/or K 1366 on the distribution of Rb86 clearance in the canine heart under selective coronary hypotension.

Effects of beta-blockers o-[2-hydroxy-3-(tert.-butylamino)-propoxy]-benzonitrile (Kö 136 1366) and propranolol, on regional Rb86 clearance in the dog myocardium under selective coronary hypotension were investigated. Selective coronary hypotension of the left circumflex artery below 50 mmHg significantly decreased the Rb86 clearance and its I/O ratio of the involved myocardium. Kö 1366 and propranolol had a general trend to decrease the regional Rb86 clearance in the non-ischaemic region and to increase it in the ischaemic region when compared with that of the control group. Therefore, the gradient of regional Rb86 clearance between the non-ischaemic and ischaemic region may decrease. However, these changes were not statistically significant. Although both Kö 1366 and propranolol groups demonstrated similar haemodynamic changes, a reduction in heart rate was larger with propranolol infusion than with Kö 1366. On the contrary, a decreasing rate in systemic arterial blood pressure was marked with Kö 1366. Epicardial ECG ST elevation was reduced after propranolol infusion, not after saline or KO 1366 infusion.


[Studies on the binding of some parasympatholytics to bovine serum albumin/measurement of relaxation times (author's transl)].

The interactions of the parasympatholytic drugs adiphenin, adiphenin H and propantheline and the structurally related compounds diphenhydramine, D-propoxyphene and methadone with bovine serum albumin (BSA) are studied by means of 1H-NMR-spectroscopy. The relaxation rates of essential protons or proton groups of the bound ligands are obtained by a plot of the observed relaxation rates, which are determined from the alterations of the line widths in the presence of BSA, versus the biopolymer concentration. From the stabilization factors R thus obtained it can be concluded that there exist defined but relatively weak associations between these drugs and BSA; both the hydrophobic aryl moieties and the ionized amino groups are involved in these interactions.


Determination of bromazepam in plasma with an internal standard by gas-liquid chromatography.

A gaschromatographic assay method is described for the determination of Ro 5-3350 (bromazepam) using Ro 5-4547 (methylbromazepam) as internal standard. After alkaline ether extraction the bromazepam and methylbromazepam obtained from sulfuric acid reextraction are hydrolyzed to ABBP and to MABBP, respectively. After neutralization, the bromo-pyridine-benzophenones are extracted with ether and dissolved in hexane after evaporation of the ether. Under the described gaschromatographic conditions it was found that MABBP and ABBP have retention times of 10.5 and 12.5 min, respectively. The limit of sensitivity is situated at 5 ng/ml of plasma. The specificity is satisfactory since the metabolite which might have interfered (hydroxy-3-bromazepam) appears only at very low concentrations in the blood. The linearity of the calibration curve was confirmed for plasma concentrations up to 100 ng/ml.


Interaction of homopyrimidazole derivatives with biopolymers. I. Binding of MZ 144, a new potent analgesic to human serum albumin.

The binding of 1,6-dimethyl-3-carbethoxy-4-oxo-6,7,8,9-tetrahydro-homopyrimidazolium-methyl-sulfate (MZ 144, Probon) to human serum albumin (HSA) has been studied in vitro. The measurements have been performed mostly by means of the classical equilibrium dialysis method. Initially several controls were run to estimate the binding of drug to Visking membrane, time required for equilibrium and to check the stability of the drug at different pH. In addition binding was studied spectrophotometrically, essentially following the method of Klotz. Treatment of binding data was based on the equation developed by Scatchard. The data obtained do not fit the single-site binding equation but could be resolved by a computer program into two sets of binding sites. At pH 7,35,4 degrees C and using a 1% HSA solution values found for binding parameters were: N1 = 0.4359, k1 = 4077 M(-1), N2 = 1.17, k2 = 424 M(-1). Protein binding is considered to have a strong effect on drug distribution only if the affinity constant for the drug, k, has a value greater than 1 times 10(4). The HSA-MZ 144 interaction is temperature dependent and pH dependent.  The percentage bound as a function of total drug concentration was calculated at pH 4.96, 7.35 and 8.5; a considerable increase was observed at pH 8.5.


Pharmacological studies of 1-(o-chlorophenyl)-2-tert.-butylaminoethanol (C-78), a new bonchodilator.

The bronchodilating effects and other related pharmacological properties of 1-(o-chlorophenyl)-2-tert.-butylaminoethanol-hydrochloride (C-78) were studied in comparison with those of isoproterenol, salbutamol and clorprenaline. C-78 was found to produce a bronchodilating effect 2--10 times stronger and considerably weaker than clorprenaline and isoproterenol, respectively. However, the bronchodilating effect of C-78 was sustained more than 10 times as long as those of isoproterenol and sulbutamol and, especially on oral administration, it was much more potent. The bronchodilating effect of C-78 seems to be due to excitation of the adrenergic beta-receptor, and was antagonized by propranolol. The positive chronotropic and inotropic effects of C-78 on an isolated atrial preparation were less than 1/1000 as potent as those of isoproterenol. C-78 also decreased the tone of smooth muscle, such as the uterus, but its affinity for the bronchial muscle is much higher than for the uterus. C-78 showed strong anti-anaphylactic action and strongly promoted the tracheal ciliary movement. Anitussive effect was also demonstrated, though this was inferior to that of codeine, however, there was no influence on bronchosecretion.


Pharmacokinetics of a new beta-adrenoceptor blocking agent, LF 17-895, in man.

The pharmacokinetics of a new potent beta-adrenoceptor blocking drug, bis-4-(2-hydroxy-3-isopropylamino-propoxy)-2-methyl indole sulphate (LF 17-895), have been studied in 5 volunteers after single oral (10 mg) and intravenous (4 mg) doses in a cross-over design. Following oral administration adsorption was rapid with peak plasma concentrations recorded after 3 h. Following the intravenous dose a biphasic decline of the plasma level curve was observed. The half-life of plasma elimination during beta-phase was 4.6 +/- 0.7 (p.o.) and 4.7 +/- 0.3 (i.v.) h, respectively. Absorption of the drug was 88.3 +/- 9.6% comparing the areas under the curve. 28.4 +/- 2.2% of the dose given i.v. was excreted in urine unchanged. When the pharmacokinetic data obtained with LF 17-895 were compared with those of pindolol, which differs only in lacking one methyl group in position 2 at the indole ring, only minor differences were seen: absorption of pindolol as well as plasma elimination were slightly faster.


[Possibilities of detecting and differentiating central nervous effects of drugs in animal experiments with special consideration of behavioral-pharmacologic methods].

Animal experimental methods suitable for the assessment of actions of drugs of the central nervous system are described with special reference to behavioral actions. The value of those methods for the prediction of central nervous actions of drugs in man are discussed.


Staphylococcal micrococcins. II. Isolation, purification and identification.

Seven strains belonging to the Micrococcaceae family and excreting substances with antibiotic activity, were grown in submerged cultures on technical scale for isolation, purification and identification of biologically active compounds. Two basic substances were isolated and classified to the micrococcin antibiotics family. The naturally occurring mixture of micrococcin M1 and M3 was called micrococcin M. This antibiotic has the formula C48H50O11N12S6 and a molecular weight of about 1160, melting point 221--224 degrees C, and optical rotation [a]20/D = + 66.6. Other antibiotically active substances produced by seven investigated strains were identified as micrococcin M or as separate compounds. Comparison with previously described micrococcin and micrococcin P has been made.


The action of some triphenylmethane dyes on yeast and erythrocyte membranes.

The action of a series of triphenylmethane dyes on the membranes of two different cell types, yeast cells and erythrocytes, have been studied. The action of the dyes on yeast cells resembles the action of other positively charged bactericides. The sequence of the activity in the dye series is completely different for yeast cells and erythrocytes. Pararosaniline and crystal violet are much more active than malachite green and brilliant green on yeast cells, whereas the reverse sequence of activities applies in erythrocytes. The carbinol form of the dyes plays an important role as regards dye interaction with erythrocytes. Transition of the dye into the carbinol form is in water extremely slow, but is greatly accelerated in the presence of an organic phase, at least for malachite green and brilliant green, but not for crystal violet and pararosaniline. This explains the different action of the two categories of dyes on erythrocytes. Experiments with pure carbinol also support the hypothesis.


[Development of an oral retard preparation of verapamil and absorption study in the anesthetized dog].

1. An oral retard preparation of verapamil (Isoptin retard) is described. The sustained release of the active compound from verapamil retard is obtained by mixing and pressing verapamil hydrochloride with sodium alginate. The proportion of active compound and physiologically indifferent additive may range between 1:1 and 1:2. 2. The determination of the in vitro release according to the half-change method shows that verapamil is completely released from the retard preparation only after 6 to 7 h; with conventional dragées more than 90% of the compound is dissolved already after 10 min. 3. In artificial gastric and intestinal juice (pH 1.4 and 7.5) similar times of release are found. 4. The in vitro results are supported by tests on the intestinal absorption in the anesthetized dog. In a ligated duodenum section verapamil is, 3 to 8 h after application, markedly more slowly released and absorbed from the retard preparation than from conventional dragées.


Drug effects on myocardial 45Ca uptake in conscious rats.

The myocardial content of 45Ca++ in conscious rats is increased by single s.c. injections of sympathomimetics. A dose dependent inhibition of this effect is achieved by simultaneous administration of calcium antagonists or beta-receptor blocking agents. The myocardial 45Ca++ content of conscious rats is increased by i.v. administration of dibutyrylcycloadenosinemonophosphate (DBcAMP). The effect of the cyclic nucleotide is suppressed only by a calcium antagonist but not by a beta-receptor blocker. The following conclusions may be drawn: 1. Calcium antagonists and beta-sympatholytics have different sites of action in the heart. 2. The lack of DBcAMP-antagonism of the beta-sympatholytics permits a simple discrimination between both types of substances. After pretreatment with sympathomimetics for 7 days (0.3 mg/kg isoprenaline s.c.), neither high doses of isoprenaline nor doses of DBcAMP and aminophylline increased the myocardial 45Ca content in rats. This effect only lasted briefly (for about two weeks); the site of its action is so far unknown.


Sedative-hypnotic properties of a new benzodiazepine in comparison with flurazepam. Pharmacological and clinical findings.

The sedative-hypnotic effects of a new benzodiazepine, 1-(2-hydroxyethyl)-3-hydroxy-7-chloro-1,3-dihydro-5-(o-fluorophenyl)-2H-1,4-benzodiazepin-2-one (SAS 643), were compared with those of flurazepam in mice and rats as well as in a double-blind clinical trial. It was found that SAS 643 has a potency 2--4 times greater than that of flurazepam while it is about one half less toxic than the latter drug. The results of the clinical trial confirm the greater activity of SAS 643 and indicate that the new benzodiazepine causes a significantly less amount of hangover than flurazepam.


[Comparison of the haemodynamic effects in man of the beta-adrenergic blocking substances Kö 1366 and practolol (author's transl)].

In 21 healthy male persons haemodynamic changes (heart rate, end-diastolic pulmonary artery pressure, training condition of heart, a so-called index of heart sufficiency and arterial blood pressure) at rest and during dynamic exercise were compared respectively before and after i.v. injection of 0.0375 mg/kg of the beta-adrenergic blocking drug K+5Ao 1366 [O-(2-hydroxy-3-(tert. butylamino)-propoxy)-benzonitril-hydrochloride], of 0.3 mg/kg practolol [4-(2-hydroxy-3-isopropylaminopropoxy)-acetanilide] and a placebo. According to the analysed parameters Kö 1366 has an essentially better beta-adrenergic blocking potency than practolol. Neither of the two substances has a negative inotropic effect or influences the arterial blood pressure.


The effect of diazepam, flunitrazepam and droperidol with an analgesic on blood pressure and heart rate in man.

The effects of i.v. diazepam (0.3 mg/kg), droperidol (5 mg) and a new benzodiazepine, 5-(a-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one (flunitrazepam, Ro 5-4200) (0.03 mg/kg) on blood pressure and heart rate was studied in 62 healthy volunteer students. Observations were made after each drug alone, after diazepam and flunitrazepam each combined with pethidine (1 mg/kg), and after droperidol combined with fentanyl (0.2 mg). The doses of the benzodiazepines were halved in those subjects given pethidine but the dose of droperidol was the same with and without fentanyl. The blood pressure was measured by auscultation and the heart rate by counting the radial pulse. The systolic and diastolic blood pressure was regularly decreased by not more than 20 and 11 mmHg, respectively. Changes in the heart rate were slight. Flunitrazepam did not have greater effects than diazepam or droperidol through the combination of flunitrazepam and pethidine seemed to induce a greater fall in systolic blood pressure than did the combination of diazepam and pethidine. None of the changes observed was clinically significant.


Phospholipase A and lysophospholipase activity of the epidermis.

Phospholipase A and lysophospholipase activities have been demonstrated in cow snout epidermis. The phospholipase A activity was dependent on Ca2+ ions and the pH for optimum activity was between 6-1 and 7-4.


Use of propranolol in dysfunctional labour.

Labour pains associated with fear and anxiety increase the blood level of catecholamines. This in turn causes dysfunctional labour due to the weak uterine contractions which follow stimulation of uterine adrenergic beta receptors. Intravenous propranolol was administered to ten primigravidae with typical dysfunctional labour. This was shortly followed by normal uterine activity and delivery without any significant maternal or fetal complications. To the best of our knowledge this is the first attempt to treat dysfunctional labour by the intravenous administration of a beta-blocking agent, and our preliminary results are encouraging.


Localization of the structural change induced in tRNA fMET (Escherichia coli) by acidic pH.

We have compared the molecular mechanism of thermal unfolding for native tRNA fMet (Escherichia coli) and the denatured species produced by annealing at pH 4.3. Relaxation kinetic measurements reveal that the transitions assigned to melting of TphiC, anticodon, and acceptor stem helices at neutral pH remain essentially unaltered at pH 4.3, but the transition corresponding to coupled melting of tertiary structure and dihydrouridine helix is greatly affected. The Tm of this region is more than 20 degrees higher at pH 4.3 and it has a larger enthalpy formation than in the native state. The transition dynamics are also considerably changed. In contrast to the native structure, tRNA fMet1 and tRNA fMet3 have similar tertiary structure stabilities at pH 4.3. We conclude that the structural difference between native and acid-denatured forms is localized in the tertiary structure-dihydrouridine helix cooperative interaction region of the molecule.


Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation.

The secondary structure of highly purified globin messenger RNA has been investigated by alkaline hydrolysis, nuclease digestion, and thermal denaturation. The thermal denaturation properties of globin messenger have been compared to poly(U), poly (A), and a synthetic random sequence RNA copolymer. From these studies it is concluded that globin mRNA contains considerable secondary structure and that the amount of helical structure is greater than that which occurs with a random sequence polyribonucleotide. Globin mRNA contains, by comparison to the secondary structures of native DNA, tRNAs, or 18S rRNA, helices with involve 55-62% of the bases or 58-68% if a correction is made for the 3'-terminal poly(A) segment. The helices of globin mRNA appear to be unique as differences in the NaCl stabilization of this RNA have been noted when compared to other naturally ooccurring and synthetic RNAs. Comparison of the hyperchromicity maxima, obtained at 260 and 280 nm for globin mRNA and 18S rRNA, indicates that the helices of the two RNAs contain similar numbers of G-C base pairs. Differential analysis of NaCl stabilization curves indicate three discrete thermally denaturable helix types in globin mRNA.


DNA synthesis in isolated HeLa cell nuclei. Optimalization of the system and characterization of the product.

DNA replication in isolated nuclei from synchronized HeLa cells has been studied in an effort to optimalize the system and characterize the product. The synthesis was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg2+. Optimum pH was about 7.8. The system was further stimulated by monovalent ions with NH4Cl and  Tris-HCl (each 65 mM) being the most effective. The four ribonucleoside triphosphates and glycerol gave a slight but very reproducible and additive stimulation. Low concentrations of spermine and spermidine (0.2-1.5 X 10(-4) M) were also slightly stimulatory (10-15%) whereas higher concentrations were inhibitory. The reaction product was DNase sensitive, and banded at 1.699 g/ml in neutral CsCl together with bulk HeLa nuclear DNA. When studied by neutral CsCl and alkaline Cs2SO4 gradients, the incorporation of [3H]TTP was mainly (more than 85%) due to further elongation of strands initiated in vivo as evidenced by BrdUrd labeling.


DNA synthesis in isolated HeLa cell nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and their subsequent ligation.

Optimum conditions for a DNA synthesizing system based on isolated nuclei have been described (Krokan, H., Bjorklid, E., and Prydz, H. (1975), Biochemistry, preceding paper in this issue) [3H]TTP-labeled nascent DNA produced during very short pulses was analyzed by centrifugation in alkaline sucrose gradients. More than 80% of the radioactivity appeared in 2-4S pieces (primary DNA pieces). It would therefore seem that the synthesis of DNA is discontinuous both in the 5' leads to 3' and in the 3' leads to 5' directions. The size of the primary DNA pieces increases from 2-4 S up to 14 S with increasing pulse length. Evidence is presented that this increase is not caused by ligation between 2-4S primary pieces. Pulse-chase experiments showed that in this nuclear system primary pieces were ligated to a product generally larger than 30 S. Evidence is also given for the initiation of primary DNA pieces in vitro.


Enzymatic activity of the second component of complement.

Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-alpha-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex G-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C1s. The action was enzymatically mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X 10(-2) mol. Cleavage of C2 by C1s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic acitivity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C42 complex, since AcGlyLysOMe competitively inhibited the C42 mediated cleavage of C3 in free solution and the C42 dependent binding of C3 to cells.


Segments of paramyosin formed by cleavage at sites of cysteine residues.

The helical muscle protein beta-paramyosin of 200,000 was treated by the general method of G. R. Jacobson et al. (1973), J. Biol. Chem. 248, 6583) for cleavage of the polypeptide chain at the site of Cys residues. The protein cleaved into two segments: CCF-1 of 140,000 daltons and CCF-2 of 60,000 daltons. The two segments were separated and some properties were compared. Circular dichroism measurements indicated that CCF-1 was completely helical and that CCF-2 was 85% in the alpha-helical form. The molecular size, resistance to pepsin digestion, stability to heat and urea, and solubility of CCF-1 were all similar to corresponding properties of a pepsin-resistant segment PPC-1 described earlier (Cowgill, R. W. (1972), Biochemistry 11, 4532). By contrast, the properties of CCF-2 were distinctly different. It was concluded that the CCF-1 segment, like the PPC-1 segment, arose from the N-terminal two-thirds of the paramyosin molecule. The CCF-2 segment from the C-terminal one-third of paramyosin had limited solubility at neutral pH that matched the low solubility of paramyosin. It was concluded that the CCF-2 region is responsible for the self-aggregating tendency of paramyosin at neutral pH and low ionic strength.


Gel filtration studies of oxyhemerythrin. I. Effects of pH on the association-dissociation equilibria.

The pH dependency of the dissociation of oxyhemerythrin has been studied by frontal gel chromatography on Sephadex G-75. The extent of dissociation is markedly pH dependent increasing below pH 6.0 and above pH 8.8. In addition, the nature of the dissociation reaction undergoes dramatic change with pH. Below pH 6.4 the rate of equilibration between species is slow relative to their time of passage through the column and they are thus resolved on chromatography. Above pH 6.6 the rate of equilibration is rapid and the various forms of hemerythrin are not resolved on migration through the column. Below pH 7.4 the dissociation is an all-or-none process with no detectable intermediates. Above pH 8.0 several intermediate species can be detected. Values for Keq and deltaG degrees are presented for various forms of oxyhemerythrin at the several pH's studied.


Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.6 kcal and +16.5 eu per mol of monomer. These values suggest that solvent adjacent to the surface of the protein undergoes rearrangement on association. Increasing ionic strength is observed to promote dissociation while decreasing the rate of attainment of equilibrium between monomers and octamers. Qualitatively similar results are observed on lowering the pH from 7.0 to 4.8, thereby linking the effects of increasing ionic strength to those of protonation of specific amino acid residues at the subunit contacts of hemerythrin. The apparent enthalpy of ionization of the amino acid residue controlling dissociation at acidic pH was found to be -1.9 to +2.1 kcal/mol. These values are consistent with a carboxyl group.


Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.


Structural role of pyridoxal 5'-phosphate, pyridoxal 5'-phosphate analogs, and other agents in the association of subunits of Bacillus alvei apotryptophanase.

Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.


Magnesium-induced self-association of calf brain tubulin. I. Stoichiometry.

The self-association of calf brain tubulin at pH 7.0 in the presence of magnesium ions has been examined by velocity sedimentation. The schlieren patterns were analyzed by methods described by Gilbert and by Cox. The observed process is best described in terms of a rapidly reversible progressive self-association of the tubulin dimer with identical chain elongation equilibrium constants, k, terminated by a ring-closing step, at degree of polymerization n = 26 +/- 2, with k26 greater than k. The end product of the polymerization reaction has a sedimentation coefficient s20,w0 k2 +/- 2 S. It is hydrodynamically equivalent to a closed ring structure observed in the electron microscope at identical conditions.


Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.


Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.


Crystallization and partial characterization of glutamate dehydrogenase from ox liver nuclei.

Glutamate dehydrogenase have been obtained in crystalline form from purified ox liver nuclear fractions. The enzyme appeared homogeneous, as judged by several electrophoretic techniques at two pH values. A comparative study with the widely known ox liver mitochondrial glutamate dehydrogenase revealed several common features, such as the allosteric effect of the nucleotides ADP and GTP, the activation at high concentrations of the cofactor NAD+, and the existence of a concentration-dependent reversible monomer-polymer(s) equilibrium. However, the two enzymes differed in many other respects. Inorganic phosphate activated nuclear glutamate dehydrogenase to a much greater extent than the mitochondrial enzyme; the substrate NH4+ showed cooperative homotropic interactions only with nuclear glutamate dehydrogenase; kinetic differences were detected with most of the reaction substrates, as well as different rates of oxidative deamination of other L-amino acids, the nuclear enzyme had a higher anodic mobility and a different chromatographic behavior on anionic exchangers. The latter evidence indicates that the glutamate dehydrogenase activity in liver is associated with two proteins which are structurally different, thus confirming the results of a separate immunological study. Preliminary evidence suggests that the enzyme in nuclei is attached to the nuclear envelope, probably the inner membrane, from which it can be solubilized by the addition of salts.


Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.


The kinetics of flavine oxidation--reduction. I. Dismutation in nonaqueous solvent.

The dismutation reactions of flavines in dimethylformanide have been investigated using the stopped-flow technique under anaerobic conditions. The ionization constants of fully reduced and oxidized tetraacetylriboflavine were measured spectrophotometrically in buffered dimethylformanide. The dismutation equilibrium of the flavine as a function of pH in dimethylformanide was roughly comparable to that reported in water and allowed the estimation of the pKa value of the flavosemiquinone. The dismutation kinetics of tetraacetylriboflavine in unbuffered dimethylformanide were investigated using the fully oxidized and reduced flavines in their neutral form at constant produce of concentrations and varying the reduction degree. The kinetics at very low reduction ratios (less than3%) were triphasic. The kinetic analysis of the initial and simultaneous formation of the anionic and neutral radicals revealed a second-order reaction. The electron transfer between the oxidized and reduced flavines was not directly coupled with prton exchange. The multiphasic time course of the reaction proceeded primarily from differences in the intrinsic rates of the direct and mixed backward dismutation reactions of the two radical species, and finally from a change in the equilibrium conditions resulting from the accumulation of anionic flavohydroquinone. An acidic-basic negative catalytic effect from the neutral flavohydroquinone appeared progressively as the reduction degree was increased. It was complete at reduction ratios higher than 30%, i.e. under conditions where the radical anion could not be observed at any reaction time. Acids with a pKa value lower than the second one of the flavosemiquinone exhibited a similar catalytic effect. These acidic-basic catalytic effects are associated with changes in the ionic state of labile intermediate dimers formed in the forward as well as in the backward direactions of the dismutation reaction. Such a transient complex revealed by the kinetic analysis could be observed directly by absorption spectroscopy in alkaline-buffered dimethylformanide. Its spectral characteristics, as well as the kinetic effects induced by substitution of the benzenoid part of the flavine, can hardly be taken into account by a quinhydrone-like structure for the intermediate dimers at any pH value. The experimental results favored a more specific interaction, possibly of covalent character, involving the benzenoid part of the isoalloxazine ring.


The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.


An affinity adsorbent containing deoxyguanosine 5'-triphosphate linked to sepharose and its use for large scale preparation of ribonucleotide reductase of Lactobacillus leichmannii.

P3-(6-(N-Trifluoracetyl)aminohex-1-yl) deoxyguanosine triphosphate has been prepared by the reaction of N-trifluoroacetyl-6-aminohexanol 1-pyrophosphate with the imidazolide of dGMP and has been characterized. This compound and the corresponding free amine, obtained by removal of the protective trigluoroacetyl group, are activators of ribonucleotide reductase of Lactobacillus leichmannii. An affinity adsorbent for the reductase, prepared by reaction of the amine derivative with CNBr-activated Sepharose, contains dGTP covalently attached through the gamma-phosphate via a six-carbon chain to the matrix. The method of synthesis of the dGTP derivative is generally applicable to the synthesis of P3-(omega-aminoalk-1-yl)nucleoside triphosphate esters for the preparation of analogous affinity adsorbents. Ribonucleotide reductase can be rapidly purified to homogeneity, on a large scale, by use of dGTP-Sepharose and conditions for optimum recovery of the enzyme have been determined. The affinity of ribonucleotide reductase and other proteins for dGTP-Sepharose is increased by either raising the ionic strength or lowering the temperature of the eluent. Elution of the enzyme from the adsorbent can be achieved between pH 5.8 and 7.3, whereas at pH 5.3 the reductase is bound extremely tightly and cannot be recovered. Ribonucleotide reductase can be eluted from the adsorbent with dGTP or urea. Elution with urea is carried out at pH 6.3, where the enzyme is stable and maximum recovery is obtained. Affinity chromatography consistently produces ribonucleotide reductase of high specific activity (170-180 units/mg). In the presence of 0.1 to 1.2 M urea or hydroxyurea, the enzyme is inhibited, but allosteric activation is unchanged. No alteration in the structure or function of the reductase was detected when the enzyme was exposed to 2.0 M urea during elution from the affinity adsorbent, but exposure for longer periods causes some inactivation.


Interpretation of the doublet at 850 and 830 cm-1 in the Raman spectra of tyrosyl residues in proteins and certain model compounds.

The doublet at 850 and 830 cm-1 in the Raman spectra of proteins containing tyrosyl residues has been examined as to its origin and the relation of its components to the environment of the phenyl ring, the state of the phenolic hydroxyl group, and the conformation of the amino acid backbone. Raman spectral studies on numerous model molecules related to tyrosine, including certain deuterium derivatives, show that the doublet is due to Fermi resonance between the ring-breathing vibration and the overtone of an out-of-plane ring-bending vibration of the para-substituted benzenes. Further examination of the effects of pH and solvents on the Fermi doublet and of the crystallographic data demonstrates that the intensity ratio of the two components depends on changes in the relative frequencies of the two vibrations. These in turn are found to be sensitive to the nature of the hydrogen bonding of the phenolic hydroxyl group of its ionization, but much less so to the environment of the phenyl ring and the conformation of the amino acid backbone. By use of the relative intensities of the doublet in model systems where the phenolic hydroxyl group is strongly hydrogen-bonded, weakly hydrogen-bonded, free or ionized, the reported Raman intensities of the doublets observed in the Raman spectra of several proteins have been interpreted. The results are compared with those obtained by other techniques.


The mechanism of formylation of five-membered cyclic phosphorodiamidates.

Cyclic N,N'-diphenylethylenephosphorodiamidates, 1, have been found to yield up to 70% of the N-formyldiphenylethylenediamines when reacted in 1 M formate buffers (50 degrees, 25% v/v dioxane-H2O, mu = 1.0). The mechanism proposed for formylation on the basis of kinetic, product ratio, and 18O-labeling studies apparently involves cyclization of a formyl phosphoramidate-amine adduct derived from formate attack on 1H+, the process probably being catalyzed by intramolecular proton transfer between the phosphoryl moiety and aniline nitrogen. An oxygen is transferred from formate to inorganic phosphate in the net reaction. The initial site of formylation with dissymmetric 1 (p-CH3O, p-Cl) was shown by 18O incorporation studies to be the p-anisidine nitrogen, which also corresponds to the isomer of thermodynamic control. Attack by formate on 1H+, therefore, results in expulsion of the more basic nitrogen. Certain mechanistic aspects of the reaction might occur in the enzyme-catalyzed ATP-dependent formylation of tetrahydrofolic acid.


Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues.

A model is proposed to account for the observation that the denaturation of small proteins apparently occurs in two kinetic phases. It is suggested that only one of these phases--the fast one--is actually an unfolding process. The slow phase is assumed to arise from the cis-trans isomerism of proline residues in the denaturated protein. From model compound data, it is shown that the expected rate for isomerism is in satisfactory agreement with the rates actually observed for protein folding. It is also shown that a simple model of protein unfolding based on the isomerism concept is very successful in accounting for many known experimental characteristics of the kinetics and thermodynamic of protein denaturation. Thus, the model is able to predict that two kinetic phases will be seen in the transition region while none are seen in the base-line regions, that both the fast and slow refolding phases lead to the native protein as the product, that the fast phase becomes the only observable phase for jumps ending far in the denatured base-line region, that most or all small proteins show a limiting low-temperature activation energy of ca. 20,000 cal, and that the relaxtion time for the slow phase seen in cytochrome c denaturation is much shorter than for all other small proteins. By utilizing "double-jump" experiments, it is shown directly that the slow phase is not part of the unfolding process but that it corresponds to a transition among two or more denatured forms which have identical spectroscopic (286.5 nm) properties. Thus, the slow relaxation is "invisible" except in the transition region where it couples to the fast unfolding equilibrium. Finally, since the present model assumes that only one of the major kinetic phases seen in denaturation reactions is concerned with the denaturation process per se, it is in agreement with numerous thermodynamic studies which show consistency with the two-state model for unfolding.


Acetylation of human serum albumin by p-nitrophenyl acetate.

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.


Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.


Coenzyme binding by native and chemically modified pig heart triphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The binding of TPNH to native and chemically modified pig heart TPN-dependent isocitrate dehydrogenase was studied by the techniques of ultrafiltration and fluorescence enhancement. A single site (per peptide chain) was found for TPNH with a dissociation constant (KD = 1.45 muM) that is quantitatively comparable to the Michaelis constant. The oxidized coenzyme, TPN+, weakens the binding of TPNH. The substrate manganous isocitrate also inhibits the binding of TPNH and, reciprocally, TPNH inhibits the binding of manganous isocitrate, suggesting that binding to the reduced coenzyme and substrate sites is mutually exclusive. Ultrafiltration experiments with carbonyl [14C]TPN+ revealed the existence of two sites with a dissociation constant (49 muM) more than ten times higher than the Michaelis constant. This observation excludes a random mechanism for isocitrate dehydrogenase or a sequential mechanism in which TPN+ binds first. Four chemically modified isocitrate dehydrogenases have been prepared: enzyme inactivated by reaction of a single methionyl residue with iodoacetate, by modification of a glutamyl residue by glycinamide (in the presence of a water soluble carbodiimide), by reaction of four cysteines successively with 5,5'-dithiobis(2-nitrobenzoic acid) and potassium cyanide, or by addition of two cysteine residues to N-ethylmaleimide. These enzymes were tested for their ability to bind TPN+, TPNH, and manganous isocitrate. In the cases of the cysteinyl and glutamyl-modified enzymes, inactivation appears to be due primarily to loss of the ability to bind the substrate manganous isocitrate. In constrast, the methionyl residue may participate in the coenzyme binding site or, more likely, may be involved in a step in catalysis subsequent to binding.


Isolation and functional characterization of hemoglobin Casper: beta106(G8) Leu replaced by Pro.

Hemoglobin Casper (beta106Leu replaced by Pro) can be separated from hemoglobin (Hb) A by isoelectric focusing on polyacrylamide gel. This abnormal hemoglobin was estimated to be 30% of teh total by both isoelectric focusing and heat lability kinetics. Its oxygen equilibrium curves indicate a high oxygen affinity, low degree of subunit interaction, and a decreased Bohr effect. Mixtures of Hb Casper and Hb A appear to bind oxygen as if no hybrid molecules exist.


Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.


Superoxide radicals and hydrogen peroxide formation in mitochondria from normal and neoplastic tissues.

Mitochondria from beef heart, Morris hepatoma 3924A and Ehrlich ascites tumor (Lettré mutant) have been studied with respect to hydrogen peroxidase and superoxide radical formation and the presence of superoxide dismutase activity (EC 1.15.1.1, superoxide:superoxide oxidoreductase). The generation of superoxide radicals and hydrogen peroxide occurs at the level of the membrane, being present also in mitochondrial fragments. Hepatoma and ascites mitochondria have little or no superoxide dismutase activity. Superoxide radicals appear to be precursors of hydrogen peroxide formation, the reaction being catalyzed by superoxide dismutase.


Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.


Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers.

Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers in the presence of rose bengal leads to a rapid loss of enzymatic activity which follows first-order kinetics. The process is pH dependent, being more rapid at higher pH. The inactivation is closely related to the destruction of histidine residues in the enzyme. It is suggested that histidine residues are largely responsible for the loss of enzymatic activity in the photooxidation. The inactivation of potato phosphorylase is retarded by substrates, whereas that of the muscle enzyme is not. The rate of photoinactivation of muscle phosphorylase b is increased with AMP, and decreased with ATP, ADP, IMP and glucose-6-P. This finding is considered to be closely related to the allosteric transition of phosphorylase.


Reactivation and aging of diphenyl phosphoryl acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) is readily in hibited by 10(-5) M diphenylphosphorochloridate even though the inhibitor hydrolyzes in a few seconds. The fluoridate is a much weaker inhibitor. The inhibited enzyme, diphenyl phosphoryl enzyme spontaneously recovers only about 50% of its activity with a half time of about 17 min at pH 7.0 and 6 min at pH 8.0. The fact that only 50% of the original activity returns is due to aging. The rates of reactivation and aging can be very greatly increased by a few percent of an organic solvent. Depending on the solvent even 1% may increase the rates by a factor of 5 or 6. The highest increase in rate was 70-fold. Quaternary NH+4 also increases the rates. Organic solvents and NH+4 also accelerate the reactivation of the much more stable diethyl phosphoryl enzyme derivative.


Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.


Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.


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.