On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.


[Transfer of glucose from UDP-glucose in microsomal membranes of rat hepatocytes (author's transl)].

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP-glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogeneous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non-ionic detergent, Triton X-100, the transfer on collagen is activated by the same detergent. Glycogen-synthase activity requires glucose 6-phosphate at an optimal concentration of 1 mM. The Km values for UDP-glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen. Characterisation of the product indicates that a protein-bound alpha1-4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose. Pre-treatment of microsomal membranes by alpha-amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.


Electron-spin resonance of nitrosyl haemoglobins: normal alpha and beta chains and mutants Hb M Iwate and Hb Zürich.

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009  amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.


Protein A from Staphylococcus aureus. Spectropolarimetric and Spectrophotometric studies.

Protein A, a cell-wall protein from Staphylococcus aureus, has been studied by spectrophotometry and spectropolarimetry. All the four tyrosines are similarily titrated with pK-a equals 10.25. Circular dichroism (CD) spectra show that the conformation of protein A is very stable over a large pH interval (0.99-11.8). The conformation is partly intact even in 6 M guanidine hydrochloride and at 80 degrees C. Protein A contains about 50% alpha-helical structure and 10-20% beta-structures. CD band maxima at 261 and 268 nm are ascribed to transitions in the phenylalanine residues and ellipticities between 275-285 nm to the tyrosine residues. Of the four tyrosines, 3.5 are perturbed by 20% polyethylene glycol, while all of them are perturbed by 20% dimethylsulfoxide. The role of the tyrosines in the reaction with the Fc-region of immunoglobulins is discussed.


Ion-pair formation as a source of enhanced reactivity of the essential thiol group of D-glyceraldehyde-3-phosphate dehydrogenase.

The reactivity and the mode of activation of the essential--SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgár, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins. Alkylations of Cys-149 of NAD-free D-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK1 equals 5.5, pK2 equals 8.2). It is concluded that there are two reactive forms of the--SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176. The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion. The two reactive forms are transformed into each other with pK2 equals 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK1 equals 5.5. In the presence of NAD, only the pH-rate profile of alkylation of D-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK1 and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex. An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.


Etonitazine-induced rigidity and its antagonism by centrally acting muscle relaxants.

Some narcotic drugs have been reported to produce increases in muscle tone in rats. In our laboratory we have found that etonitazine produces a 'lead-pipe' rigidity of the trunk and limb musculature. The reported studies were conducted to characterize etonitazine-induced rigidity more fully, to compare the degree of rigidity with that produced by morphine, codeine and methadone, and to assess the sensitivity of this rigidity to centrally acting muscle relaxants. Of the 4 narcotics tested, etonitazine was far more potent than methadone or morphine for producing rigidity; codeine did not produce peak rigidity comparable to the other 3 narcotics. Etonitazine-induced rigidity occurs at a supraspinal level since the effect was prevented by spinal transsection. Etonitazine-induced rigidity was prevented by the narcotic antagonists cyclazocine, pentazocine and naloxone but not by the serotonin depletor, p-chlorophenylalanine. Etonitazine-induced rigidity was antagonized by centrally acting muscle relaxants, including diazepam, methocarbamol, carisoprodol and zoxazolamine; in agreement with their relative clinical muscle relaxant potencies, diazepam is the most potent antagonist of etonitazine-induced rigidity.


Reactivity of isolated bovine mesenteric and hepatic veins to vasoactive agents and specific antigen.

In an attempt to investigate the possible contribution of the mesenteric and hepatic vasculature to the haemodynamic changes occurring during bovine systemic anaphylaxis, the pharmacological reactivity of isolated bovine mesenteric and hepatic veins was studied. Both mesenteric and hepatic veins contracted in the presence of histamine, 5-hydroxytryptamine (5HT), acetylcholine, dopamine and noradrenaline, and relaxed in the presence of isoprenaline. The mesenteric veins contracted in the presence of concentrations of isoprenaline greater than 1 mug/ml. By the use of antagonists, the receptor populations of bovine mesenteric and hepatic veins were shown to be similar to those of many other vascular preparations. Hepatic veins isolated from calves previously sensitised to horse serum contracted forcefully in the presence of horse plasma (the Schultz-Dale phenomenon), whereas mesenteric veins isolated from the same animals did not. Hepatic veins may be implicated in the haemodynamics of bovine systemic anaphylaxis.


Adrenergic lipolysis in human adipose tissue in vitro.

In human adipose tissue in vitro, dose-response curves of lipolytic agents in releasing free fatty acids and glycerol into an albumine-containing medium were followed. Norepinephrine and adrenaline produced about 20% of the maximal effect of isoproterenol, isopropylnorsynephrine about 40%, theophylline more than 100%. Found pD2 values were approximately 7.4 for isoproterenol and norepinephrine, approximately 5.9 for isopropylnorsynephrine. Phentolamine (1 times 10-5 M) elevated the maximal norepinephrine effects up to the isoproterenol maxima. Phenoxybenzamine (1 times 10-5 M) had significantly lower potentiating effects. Phenylephrine depressed lipolytic actions of isoproterenol (1 times 10-6 M) showing the same pI2 value 3.0 as well in human as in rat adipose tissue and exerted per se weak lipolytic effects also. It is concluded neither potentiating actions of alpha-blockers nor depressing effects of phenylephrine in human adipose tissue seem related to affecting adrenergic alpha-receptors. The existence of antilipolytic actions of adrenergic alpha-receptors is questioned.


Studies on the temperature-dependent sensitivity of mouse atria to adrenergic drugs.

The sensitivity of mouse atria (chronotropic response) to beta-adrenergic receptor antagonists was examined at 37 and 26 degrees C under various experimental conditions. When isoproterenol was used as agonist, at 37 degrees C, the pA2 value (from pA2 plots) for propranolol was 8.86 and the slope of the regression line was 0.54. At 26 degrees C, the pA2 value for propranolol was 9.2 and the slope was 0.7. In the presence of tropolone, 10-5 M, the values for pA2 and slope of the regression lines were, respectively, 9.0 and 0.90 at 37 degrees C and 9.17 and 0.98 at 26 degrees C. In other words, decreasing antagonism was prevented by low temperature and tropolone. Isoproterenol was potentiated by tropolone and low temperature, but the effects of low temperature were reduced by tropolone. With sotalol as antagonist, there was greated blockade (dose ratios) of the effects of isoproterenol at 26 than at 37 degrees C. When nylidrin, a non-catecholamine, was used as agonist there was no temperature-dependent sensitivity to sotalol. Furthermore, nylidrin was not potentiated by low temperature. The data show that the observed degree of beta-receptor antagonism can be altered by changing the bath temperature and suggest that this effect is related to COMT activity or an influence of agonist disposition in the tissue.


The central modulatory effect of clonidine on the cardiodepressor reflex after suppression of synthesis and storage of noradrenaline.

Rats were decerebrated and treated with a beta-adrenoceptor blocker (toliprolol, Kö 592, 5 mg/kg). Increases of blood pressure were repeatedly elicited by i.v. injection of angiotensin (0.03-0. 125 mug/kg) ant eh accompanying reflex bradycardia was measured. The vagally mediated reflex bradycardia was significantly increased by clonidine, 3o mug/kg i.v., in non-pretreated animals as well as after pretreatment with reserpine (7.5 mg/kg s.c., 20 hr) and alpha-methyl-p-tyrosine-methylester (250 mg/kg i.p., 5 hr), separately or in combination. The facilitatory effect of clonidine was antagonized in all groups by i.v. injection of the alpha-adrenoceptor blocking drugs, phentolamine (5 mg/kg) and piperoxan (1 mg/kg). In the control periods before clonidine, reflex bradycardia was similar in pretreated animals to that in animals without pretreatment. It was concluded therefore that catecholamines have no essential transmitter functions in the cardiodepressor reflex loop. Therefore, the action of clonidine on cardiovascular centers in the medulla is independent of endogenous noradrenaline storage and synthesis. A direct effect is assumed on central alpha-adrenoceptors, which have a modulatory 'effector' function on the cardiodepressor reflex.


Several recent experiments demonstrate the presence of an essential negatively charged acid group within sodium channels. Sodium permeability titrates away at low pH as if controlled by an acid with a voltage-dependent apparent pKa in the range between 5 and 6. The alkali ion permeability sequence of the channel is best explained by interactions between the cations and a strong negative charge in the channel. Block of sodium currents by a variety of metal and organic cations again points to a cation-coordinating site in the channel. The "blocking cations" and protons also oppose the bindings of tetrodotoxin and saxitoxin. The negative charge in the channel seems to be essential in selecting appropriate cations and in lowering their activation energy for permeation. The same charge seems to form part of the toxin receptor. At present this charged group is the chemical group known to be associated with sodium channels.--Hille B. An essential ionized acid group in sodium channels.


Non-specific decrease of cell-mediated immunity in female mice during syngeneic and allogeneic pregnancy.

Cell-mediated immunity is non-specifically reduced in the second half of H-2 incompatible pregnancy compared with syngeneic mating. Significant difference between syngeneic and allogeneic mated female mice was obtained using two independent tests: contact sensitivity to oxazolone (P smaller than 0.001) and local graft-versus-host reaction (P smaller than 0.01).


Red cell abnormalities associated with sulphasalazine maintenance therapy for ulcerative colitis.

Of 52 patients receiving a mean dose of 2.5g sulphasalazine/day as maintenance therapy for ulcerative colitis, 35 were found to have one or more drug-induced red cell abnormalities, which were not found in 50 normal controls or in 10 colitics not receiving sulphasalazine. Twenty-three of the treated patients had contracted red cells, an abnormality that is thought to result in mild haemolysis. Red cell contraction was related to the dose of sulphasalazine (P smaller than 0.01), the serum total sulphapyridine level (P smaller than 0.001), and acetylator status. Eleven of the treated patients had a macrocytosis, 21 had elevated levels of methaemoglobin, and one had Heinz bodies. A dose of 1.5 g sulphasalazine/day was not associated with red cell contraction, and is suggested as a safer maintenance dose for the asymptomatic colitic.


In vitro cytotoxicity of anti-theta (Thy-1) antibodies combined with chlorambucil.

Two methods are described for linking chlorambucil to antibodies. At low pH an association is formed which is not stable under physiological conditions and the cytotoxicity is the result of the independent action of chlorambucil and antibody. At pH 8 a more stable linkage is formed and this complex shows specific toxicity for cells carrying the appropriate antigen. Uncombined chlorambucil and antibody are more cytotoxic that chlorambucil alone, especially if the cells are exposed first to the chlorambucil.


Reactions of purified hemagglutinating antigens of flaviviruses with 19S and 7S antibodies.

The rapidly sedimenting hemagglutinin (RHA) representing the purified virion and the slower-sedimenting hemagglutinin (SHA) of several flaviviruses were separated and used in hemagglutination inhibition tests with the 19S (immunoglobulin M) and 7S (immunoglobulin G) immunoglobulin fractions of rabbit antisera, prepared against purified viral antigens or against crude virus pools. The antibody specificity in tests with RHA was identical to the specificity in those employing SHA. 7S antibody cross-reacted broadly with all flavivirus antigens, whereas 19S antibodies were relatively specific in cross-reactions among flaviviruses (RHA or SHA). SHA was consistently inhibited by antibody to a greater extent than RHA. Anti-envelope protein, anti-RHA antibodies and anti-SHA antibodies were unable to discriminate between RHA and SHA. It was concluded that the relative amounts of RHA or SHA in crude hemagglutinin preparations have no influence on the result of hemagglutination inhibition tests with flaviviruses.


T lymphocyte function as the principal target of lymphocytic choriomeningitis virus-induced immunosuppression.

Plaque-forming cell responses against sheep erythrocytes, Escherichia coli lipopolysaccharide, pneumococcal polysaccharide, and polyvinylpyrrolidone were examined in mice infected with lymphocytic choriomeningitis virus.  A 92 to 96 percent reduction of the thymus-dependent anti-sheep erythrocyte responses was observed 2 to 4 weeks after infection.  However, the thymus-independent responses against the three other antigens were close to normal at all stages of the infetion.  Studies on allograft immunity of infected C3H mice against DBA/2 mastocytoma cells revealed a severe suppression of the T cell-mediated cytotoxic response which was temporally related to the impaired humoral responsiveness against sheep erythrocytes.  The capacity of spleen cells from infected mice to restore immune responsiveness of lethally irradiated recipients against sheep erythrocytes was significantly reduced.  The adoptive responses, however, were clearly improved when normal thymus cells were added to the inferior spleen cells.  Moreover, it appeared that the spleen cells from immunosuppressed donor mice could not confer suppression to normal lymphoid cells.  The presented findings are consistent with the assumption that a numeric deficiency of T cells, or cells belonging to some T cell subpopulation, is the primary cause of lymphocytic choriomeningitis virus-induced immunosuppression.


Reactivation of latent Herpes simplex virus after pneumococcal pneumonia in mice.

In attempts to reactivate latent herpes simplex virus, we instilled Diplococcus pneumoniae intratracheally into mice harboring latent infections in sacrosciatic spinal ganglia. All mice developed a severe pneumonia within 24 h and were given penicillin therapy. Representative mice that survived the penumonia were sacrificed at daily intervals, and appropriate tissues were examined for evidence of viral reactivation. Herpes simplex virus was reactivated in the ganglia and appeared to travel both proximally and distally in associated nerve trunks. Clinically apparent disease due to the virus was not detected in any mice.


Chemical modulation of diphtheria toxin action on cultured mammalian cells.

Ammonium chloride (4 times 10-3 M) rendered HEp-2 monolayers completely insensitive to the action of diphtheria toxin, as measured by de novo protein synthesis. Total protection was observed even with large amounts of toxin (400 minimum lethal doses/ml). Ammonium chloride did not reduce toxicity by direct action on the protein, nor did it prevent the adsorption of toxin to the cell membrane. Although the ammonium salt did not block the initial interaction between cell and toxin, it did maintain the toxin at a site amenable to neutralization with antitoxin. Surface-adsorbed toxin was inactivated by cellular enzymes or alternatively was desorbed from the membrane during a 12-h incubation in the presence of ammonium chloride. In addition, ammonium chloride provided protection to both toxin-sensitive guinea pig peritoneal macrophages and a partially toxin-resistant strain of HEp-2 cells. Sodium arsenite was effective in protecting cell monolayers from the action of diphtheria toxin; unlike ammonium chloride, its action was not dependent upon continued incubation with cells during exposure to toxin. Inhibitors of energy metabolism abolished toxin action either totally (sodium fluoride) or partially (dinitrophenol and sodium cyanide). Inhibitors of cellular proteases, on the other hand, did not modify toxin activity. The ability of several modifiers of membrane function to alter expression of toxicity for HEp-2 cells was also examined. One compound known to enhance endocytic activity, Tuftsin, had no effect, whereas poly-L-ornithine provided partial protection. Of the two compounds known to alter membrane fluidity, cytochalasin B provided partial protection for HEp-2 cell cultures, whereas colchicine had no effect. Agents that bind to sulfhydryl groups on the cell surface had no apparent effect on toxicity, suggesting that the initial toxin-cell interaction does not involve sulfhydryl groups. Those compounds that provide virtually full protection against the action of diphtheria toxic on cell monolayers (i.e., ammonium chloride, sodium fluoride, and sodium arsenite) had no inhibitory effect on the in vitro enzyme activity associated with fragment A of the toxin.


Changes in blood pH in rats after infection with Streptococcus pneumoniae.

Acid-base alterations in Streptococcus pneumoniae infection were studied in 80 male albino rats. Hematocrit and concentrations of plasma electrolytes, glucose, and total protein were also measured. At 3-h intervals throughout a 27-h study, four control and four infected rats were anesthetized with ether, and blood samples were taken. Arterial blood pH, Po2, and hematocrit increased in the infected group, whereas arterial Pco2, HCO3-, and venous Po2 decreased. Plasma K+ concentration increased slightly and glucose levels decreased in the infected rats as the sepsis progressed. No significant changes were observed in venous blood pH, HCO3-, and Pco2. Plasma Na+, Cl-, and total protein remained unchanged. The increase in arterial blood pH and decrease in arterial Pco2 and HCO3- indicated respiratory alkalosis, which was present in rats infected with S. pneumoniae.


Partial purification and characterization of RTG-2 fish cell interferon.

Interferon produced by rainbow trout gonadal cells (RTG-2) was partially purified. The physical, chemical, and biological properties of this in vitro produced fish cell interferon were studied. Purification was achieved by ultracentrifugation, molecular sieve gel chromatography, ion exchange chromatography, and polyacrylamide gel electrophoresis. The isoelectric point of RTG-2 interferon, as determined by CM-Sephadex (C-50) chromatography, was 7.1. Filtration through Sephadex G-150 showed that RTG-2 interferon had a molecular weight of 94,000. The partially purified material was not sedimented at 105,000 times g for 2 h at 4 C. The fish cell interferon was non-dialyzable and exhibited heat and pH stability. The partially purified material was inactivated by treatment with trypsin or 2-mercaptoethanol, but was resistant to treatment with deoxyribonuclease or ribonuclease. RTG-2 interferon which was induced by infectious pancreatic necrosis virus exhibited antiviral activity against challenge with infectious hematopoietic necrosis virus or infectious pancreatic necrosis virus. Partially purified RTG-2 interferon exhibited greater species specificity than the crude material.


Correlation between mature and immature thymocytes. Immature thymocyte potentiating factor (IMPF) produced by mature thymocytes.

Low-dense mature thymocytes (MT) and high-dense immature thymocytes (IMT) were investigated. Immature thymocyte-potentiating factor (IMPF) was found in the supernatant from MT stimulated by Con A. On the other hand, suppressing activity was present in the supernatant from the unstimulated IMT. IMPF was different from Con A itself. Sephadex G-100 gel chromatography of IMPF revealed that the activity was recovered at the position between bovine serum albumin and ovalbumin.


Impairment of mitochondrial respiratory control by activated lymphocytes.

Mitochondria isolated from rat livers were used as a source of antigens capable of producing in vitro activation of lymphocytes that had previously been sensitized in vivo with the same antigen. Such lymphocytes were shown to have an injurious effect on target mitochondria, as was demonstrated by a new biochemical approach based on the study of the inhibition by these cells of mitochondrial respiratory control. A somewhat milder injurious capacity was also observed in normal and sensitized lymphocytes activated in vitro with the mitogen PHA. The lateration of the respiratory control of mitochondria by lymphocytes correlated with an increase in DNA synthesis in these cells.


Some properties of cathepsins chemically fixed to carriers.

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.


Synthesis and charge-transfer properties of two acth analogues containing pentamethylphenylalanine in position 9.

In order to investigate the possible role of the Trp residue in ACTH as a change-transfer donor in the activation of ACTH receptors, two ACTH analogues (beta-corticotrophins (I-24) with L-Ser1 and D-Ser1, respectively) containing pentamethylphenylalanine instead of Trp have been synthesized. In these syntheses a new, alkaline-labile, amino-protecting group, the methylsulphonylethyloxycarbonyl group, was employed. The association constants of ACTH (I-24) and (Pmp9)-ACTH (I-24) with the water-soluble acceptor paraquat, were nearly equal.


A study of the anticholinergic and antispasmodic activity of oxybutynin (Ditropan) on rabbit detrusor.

Experiments have been conducted which show that while the tertiary amine oxybutynin has only moderate in vitro anticholinergic and antihistaminic activity, it is a potent inhibitor of barium chloride-induced spasms of rabbit detrusor muscle. Oxybutynin exerts 1/13 the anticholinergic activity of propantheline and 1/4 that of atropine, but produces approximately 2x the inhibition of barium chloride-induced spasm than propantheline and 10x the inhibition produced by atropine. In sum, oxybutynin possesses a notable antispasmodic activity distinct from its anticholinergic activity and stronger than that of atropine, propantheline, or methantheline, which may prove useful in the treatment of smooth muscle hypermotility.


Contrast media used in cystourethrography. Experimental evaluation.

The effects of five iodinated contrast media and of physiologic saline on bladder capacity were studied. It was found that a significantly greater volume of sodium chloride was tolerated than of Cysto-Conray, Conray 30. Hypaque meglumine 30, Hypaque sodium, and Dimer X. Dimer X was tolerated in the smallest volume. The reason for the difference in volume tolerated is not known. The bladder mucosa showed dehydration changes 6 hr after saline cystogram with no change at 24 hr, 48 hr, 7 days, and 3 months. The contrast media produced dehydration and hydropic and desquamative changes in the bladder mucosa with at least one of these changes persisting up to 3 months. There was no significant difference in the severity of the changes produced by the various contrast media. No evidence of inflammatory changes was seen in the bladder mucosa or wall or in the ureter with any of the agents.


A genetical study of DDT resistance in the mosquito Aedes aegypti.

Crosses have been carried out to determine the relationship between adult DDT resistance and the three linkage groups of the mosquito Aedes aegypti. Two linkage groups were implicated in the control of DDT resistance. In the Bangkok-HR strain resistance derived mainly from linkage group III, probably with the maor effect from the gene R-DDT2. When resistance was transferred into a susceptible background, by outcrossing Bangkok-HR to strain 64 and reselecting, resistance in the resulting Bangkok-MR strain came from both linkage groups II and III.


Constituents of human muscle in isometric fatigue.

Three subjects performed five successive isometric contractions to fatigue; the tension in any one experiment was constant at tensions varying from 20 to 80% of the maximal voluntary contraction (MVC). The interval between contractions was held constant at 11 min. Muscle biopsy specimens were obtained at the start of the experiment, after the first, fourth, and fifth, and before the second and fifth of the successive contractions. The concentrations of ATP, CP, glycogen, and lactate were measured in each sample of muscle. Changes in ATP and glycogen were insufficient to be held accountable for the development of isometric fatigue. Changes in CP and lactate were large after fatigue at intermediate tensions, but those of CP were considered unlikely to be responsible for the fatigue. At tensions of 30-50% MVC the increase in lactate could be responsible for fatigue either directly or by indirect changes in pH; at higher and lower tensions the possibility that lactate is directly implicated in the development of fatigue seems remote.


Pulmonary ventilation, blood gases, and blood pH after training of the arms or the legs.

In two groups of young healthy subjects who performed arm training (N = 5) and leg training (N = 5), respectively, the respiratory adaptation to submaximal exercise with trained and nontrained muscle groups was compared by measurement of the ventilatory equivalent (Ve/Vo2, pH, and blood gases (Pco2, Po2, and So2) in arterial blood and in venous blood from exercising extremities. After training Ve/Vo2 was significantly reduced during exercise with trained muscles, but unchanged during exercise with nontrained muscles. The reduction in Ve/Vo2 was closely related to a less pronounced increase in heart rate and in arterial lactate content, but showed no quantitative correlation to changes in arterial adaptations in trained muscles are mainly responsible for the reduction in Ve/Vo2. After training during exercise with trained as well as nontrained muscles a shift to the right of the blood oxygen dissociation curve occurred as extremities was lower while corresponding Po2 was higher.


Tissue oxygenation and splenic erythropoiesis during chronic hypoxia and hypercapnia.

Tissue (gas pocket) oxygen levels and erythropoietic activity were monitored in groups of rats chronically exposed to hypoxia (70 Torr PIO2), hypercapnia (60 Torr PICO2), or a combination of the two conditions. Arterial gas tensions and pH were also measured. Overall condition of the animals was assessed by comparison of growth rates with pair-fed controls. Hypoxic-hypercapnic pocket PO2 values (24-40 Torr) were similar to normoxic-normocapnic values (28-37 Torr), but greater than in hypoxia-normocapnia, and less than in normoxia-hypercapnia. Erythropoietic activity during hypoxia-hypercapnia ceased and the rats had a growth rate significantly below that of other groups. While chronic CO2 does increase tissue (pocket) oxygenation to near normal levels, probably due to increased ventilation and subsequently PaO2, the hypoxic-hypercapnic rats evidenced greater detrimental effects than did rats in hypoxic or hypercapnic environments.


Characterization of the electron transport system in Brucella abortus.

The electron transport system in Brucella abortus has been characterized. Spectral studies of membrane preparations have indicated the presence of cytochromes a + a3 (maxima at 612 nm), cytochrome b (maxima at 560, 530, and 428 nm), cytochrome c (maxima at 552 and 522 nm), cytochrome o (maxima of carbon monoxide complex at 418 nm), and flavoproteins (minimum at 582 and 450 nm). Cytochromes a + a3 appeared only after cells had reached late log phase, possibly due to lowered oxygen tension in the medium. Dehydrogenases were shown to be present for D-erythritol 1-phosphate, L-lactate, reduced nicotinamide adenine dinucleotide, and succinate. All of the above substrates reduced the electron transport chain and at least some of the flavoproteins, indicating similar pathways of electron transport. N-ethylmaleimide, p-chloromercuribenzoate, and KCN were the only electron transport inhibitors that blocked electron transport by 100%. The system seemed to be uniquely resistant to other electron transport inhibitors.


Relationship between calcium and uroinic acids in the encystment of Azotobacter vinelandii.

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.


Psychrophilic, mesophilic, and thermophilic triosephosphate isomerases from three clostridial species.

Triosephosphate isomerase was purified to homogeneity as judged by analytical gel electrophoresis from clostridium sp. strain 69, clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative isoelectric point values of these three enzymes.


Membrane lipoteichoic acid is not a precursor to wall teichoic acid in pneumococci.

The distribution of a pulse of teichoic acid-specific radiolabel between wall and membrane teichoic acids in pneumococci was constant over a subsequent chase period, suggesting that wall and membrane teichoic acids are biosynthesized simultaneously and independently.


Growth of Pseudomonas C on C1 compounds: enzyme activites in extracts of Pseudomonas C cells grown on methanol, formaldehyde, and formate as sole carbon sources.

Pseudomonas C can grow on methanol, formaldehyde, or formate as sole carbon source. It is proposed that the assimilation of carbon by Pseudomonas C grown on different C1 growth substrates proceeds via one of two metabolic pathways, the serine pathway or the allulose pathway (the ribose phosphate cycle of formaldehyde fixation). This contention is based on the distribution of two key enzymes, each of which appears to be specifically involved in one of the assimilation pathways, glycerate dehydrogenase (serine pathway) and hexose phosphate synthetase (allulose pathway). The assimilation of methanol in Pseudomonas C cells appears to occur via the allulose pathway, whereas the utilization of formaldehyde or formate in cells grown on formaldehyde or formate as sole carbon sources appears by the serine pathway. When methanol is present together with formaldehyde or formate in the growth medium, the formaldehyde or formate is utilized by the allulose pathway.


Nanosecond spectroscopy of retinol.

Nanosecond fluorescence spectroscopy was used to study the unique binding site of the retinol-binding protein (RBP) from human serum. At pH 7.4, the binding of retinol to RBP caused the following spectroscopic changes in the ligand: (a) an enhancement of the fluorescence decay time (gamma = 8 ns); and (b) an increase in the emission anisotropy (A = 0.29). Retinol in hexane has a fluorescent decay time of 4.2 ns and a low emission anisotropy (A = 0.02). The increase in the fluorescence decay time of bound retinol is not due to dielectric relaxation effects of polar groups, since nanosecond time-resolved emission spectra of either retinol in glycerol or retinol bound to RBP, failed to show any time-dependent shifts in emission maxima during the time period investigated 0 to 30 ns. The degree of rotational mobility of bound retinol was investigated by time emission anisotropy measurements. The observed rotational correlation time (theta = 7.2 ns) is consistent with a rigid compact macromolecule of 21,000 molecular weight.


Characterization of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

The phosphorylation of two purified vaccinia virus proteins (Acceptors I and II) by a protein kinase isolated from vaccinia virus cores has been studied. Phosphorylation of viral acceptor proteins by the purified enzyme was dependent on the presence of ATP, Mg2+, and protamine or other basic proteins, and was maximal at alkaline pH values. Cyclic mononucleotides did not stimulate the vaccinia protein kinase under a variety of conditions. Protamine, however, was shown to function as an enzyme activator. In its presence, the purified vaccinia protein kinase phosphorylated mainly serine residues in Acceptor I, and predominantly threonine residues in Acceptor II. Phosphorylation of protamine accounted for less than 1% of the total 23P incorporation. Tryptic peptide maps prepared from 32P-labeled Acceptors I and II demonstrated that they contained different labeled peptide sequences and were, therefore, distinct protein species. From additional studies on both purified and virus-associated protein kinase it was concluded that various proteins affected the protein kinase reaction in one of three ways. One class of proteins served as phosphate acceptors, but only when another activator protein was present. A second class consisted of proteins that were strong activators but poor phosphate acceptors. The third class contained proteins that were fair phosphate acceptors, but which also activated the phosphorylation of other acceptor proteins.


Purification of membrane proteins from Acholeplasma laidlawii by agarose suspension electrophoresis in Tween 20 and polyacrylamide and dextran gel electrophoresis in detergent-free media.

Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay.


Effects of metals and nucleotides on the inactivation of sea urchin sperm guanylate cyclase by heat and N-ethylmaleimide.

Preincubation of sea urchin sperm guanylate cyclase at 35, 37, 40, or 43 degrees resultedin inactivation. Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+ were 123, 361, 5.5, 692, 984, 335, 79, and 47 muM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, minus 0.1, 0.30, 0.59, 0.66, 0.28, and 0.42 for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+, respectively. GTP, MgGTP, and SrGTP protected the enzyme only slightly against heat inactivation, but CaGTP and MnGTP protected substantially. Neither CaGTP nor MnGTP protected maximally, however, unless the metal concentration exceeded that of GTP. At fixed free Mn2+ or free Ca2+ concentrations, protection curves as a function of MnGTP or CaGTP appeared to be sigmoidal, suggesting multiple nucleotide binding sites. MnATP also protected against heat, but CaATP was virtually ineffective. Sea urchin sperm guanylate cyclase was inactivated by N-ethylmaleimide; CaGTP and MnATP were effective protectants with estimated binary enzyme-Me2+ nucleoside triphosphate dissociation constants of 40 and 170 muM, respectively. MnGTP protected only slightly or not at all against N-ethylmaleimide. These results suggest that: (a) sea urchin sperm guanylate cyclase binds free metal, (b) the binding of free metal is required for protection by nucleotides, and (c) the enzyme contains multiple nucleotide binding sites.


Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..


Acid-base catalysis in the yeast alcohol dehydrogenase reaction.

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-1,1-h2 and -1,1-d2 by NAD+ was found to be characterized by large deuterium isotope effects (kH/kD = 4.1 plus or minus 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride trahsfer step in this pH range; a plot of kCAT versus pH could be fit to a theoretical titration curve, pK = 8.25, where kCAT increases with increasing pH. The Michaelis constnat for p-CH3 benzyl alcohol was independent of pH. The reduction of p-CH3 benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) cound not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kCAT was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 plus or minus 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and exxentially invariant (kH/kD = 2.O plus or minus 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transger step for this substrate; a plot of kCAT/K'b (where K'b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for p-CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.


Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.


Formation and excretion of pyrrole-2-carboxylic acid. Whole animal and enzyme studies in the rat.

A corrected method for the measurement of pyrrole-2-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings implicated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyrrole-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studied showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxy-proline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate in urine. The capacity of homogenates of guinea pig kidney and human kidney to carry out oxidation of hydroxy-L-proline to pyrrole-2-carboxylate was much less than that of rat kidney, consistent with the lower levels of urinary pyrrole-2-carboxylate in these species. Experiments designed to examine the modest increase of pyrrole-2-carboxylate excretion after proline loads led to new observations on tissue levels of hydroxy-L-proline following proline administration and on the inhibition by L-proline of hydroxy-L-proline oxidase.


Xanthosine-5'-phosphate amidotransferase from Escherichia coli.

The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate. This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase. The so-called XMP aminase from the E. coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase. These data demonstrate that this enzyme from E. coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.


Magnetic resonance study of exchangeable protons in human carbonic anhydrases.

A titratable exchangeable proton resonance assignable to a histidine imidazole ring N--H proton is observed approximately minus 15 ppm downfield from tetramethylsilane. The chemical shift of this resonance is affected by sulfonamide and anion inhibitors, and by removal of zinc or replacement of zinc by cobalt, indicating that the proton is located at or near the active site. The pH dependence of the chemical shift of this resonance, which is abolished by inhibitors, reflects the titration of a group with a pK-a of 7.3 in human carbonic anhydrase B and smaller than or equal to 7.1 in human carbonic anhydrase C. These pK-a values are interpreted to be due to the ionization of a neutral imidazole to form the imidazolate anion coordinated to zinc. A mechanism for enzymatic catalysis involving reversible deprotonation and coordination of a histidine to the metal is consistent with these studies.


Conversion of pepsinogen to pepsin. Further evidence for intramolecular and pepsin-catalyzed activation.

Exposure of pepsinogen to acid for less than 2 min yields a product with proteolytic activity. This activity is due to intramolecular and intermolecular formation of pepsin from pepsinogen. We find no evidence for intermolecular proteolytic activity in the zymogen. These conclusions are based upon two sets of experiments. First, chemical cleavage of pepsinogen during short activation is demonstrated by quantitative analysis of the NH2-terminal 2 residues of the pepsin and pepsinogen in an activation mixture. In addition, quantitative NH2-terminal analyses after activation under different conditions confirm our previous inference that the product of unimolecular pepsinogen activation is homogeneous whereas bimolecular activation produces a pepsin product with a variety of NH2 termini. Second, spectral changes which occur upon acidification of a pepsinogen solution and are reversed by neutralization are shown to be consistent with the chemical cleavage of pepsinogen during acidification. The first order rate constant for pepsinogen activation, calculated from these spectral experiments, agrees well with the value we had determined previously.


Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.


Membrane-bound neuraminidases of rat liver. Neuraminidase activity in Golgi apparatus.

The bulk (60 to 65%) of the neuraminidase activity present in rat liver homogenates was found in the M + L (mitochondria plus lysosomes) fraction, The patterns of subcellular distribution were essentially identical whether disialogangliosides or neuramin-lactose (2 yields 3') were utilized as substrates. A new neuraminidase, which hydrolyzes sialyl trisaccharides but which does not act upon glycoproteins and gangliosides, was detected in Golgi apparatus. Unlike the other particulate neuraminidases of rat liver, the Golgi enzyme is stimulated by prior incubation and by the addition of Ca2+ or Zn2+ at 1 mM concentration. Although plasma membrane-rich fractions are often contaminated by Golgi membranes the marked differences in their enzymic properties allowed a clear distinction between the neuraminidases present in these two types of membranes.


Solvent-temperature perturbations of ionizable groups as a tool for the investigation of the active site of enzymes.

Co-solvent and temperature effects on the pK of histidine (imidazolium) residue 46 of trypsin, as well as of weak electrolytes (buffers), which have been reported in two preceding papers, can be satisfactorily explained in terms of enthalpy-entropy compensation patterns. Such patterns have been generated for various mixed solvents between 20 degrees and minus 20 degrees and minus 50 degrees. Under these conditions compensation temperature, T-c, is strongly dependent on the nature of the ionizable group studied: 240 plus or minus 10 K for neutral acids and 310 plus or minus 5 K for cationic acids. This work focuses on the possibilities offered and on the problems raised by the use of this methodology as a tool in the investigation of the active site of enzymes. Furthermore, it is shown in the case of histidine residue 46 of trypsin that the co-solvent effect vanishes at the compensation temperature, a result of great practical significance if applicable to any ionizable group at the active site of enzymes.


Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.


Spectral studies of conformational changes induced in beta-glucuronidase by saccharo-1,4-lactone.

Ultraviolet difference spectroscopy has been used to study the binding of the transition state analog saccharo-1,4-lactone to purified rat preputial gland beta-glucuronidase. At pH 4.5 (the pH optimum), the inhibitor induces a difference spectrum indicative of a change in the environment of tryptophyl residues. Based on the magnitude of the induced difference spectrum as a quantitative measure of inhibitor binding, a titration curve for saccharo-1,4-lactone was obtained. A Scatchard plot of the titration data indicates that 4 molecules of inhibitor bind to the enzyme tetramer at a K-I of 4 times 10-7 M. The inhibitor also induces a similar difference spectrum at pH 7.5, although the binding is considerably weaker at this pH than at pH 4.5. When the native enzyme at pH 4.5 is compared with the native enzyme at pH 7.5, a difference spectrum, distinct from that of the binding of saccharo-1,4-lactone, is observed, indicating that the enzyme exists in different conformations at these pH values. The indication that tryptophyl residues are perturbed upon binding of saccharo-1,4-lactone was supported by studies carried out with N-bromosuccinimide. At pH 4.3, this reagent was found to oxidize 6 tryptophyl residues in the native enzyme but only three in the saccharo-1,4-lactone-inhibited enzyme. A spectrophotometric titration of the enzyme indicated that of the 33 tyrosyl residues per subunit, only 5 to 6 ionize at the pK expected for free phenolic groups.


Thioredoxin reductase-mediated hydrogen transfer from Escherichia coli thioredoxin-(SH)2 to phage T4 thioredoxin-S2.

Thioredoxin from Escherichia coli B and phage T4-infected E. coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E. coli and T4-induced ribonucleotide reductase, respectively. The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E. coli thioredoxin reductase. Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence. By using the spectrofluorimetric properties of T4 thioredoxin and E. coli thioredoxin as markers for their oxidized and reduced forms we have shown that E. coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E. coli thioredoxin-S2 and T4 thioredoxin-(SH)2. This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E. coli thioredoxin.


Prostaglandin 9-hydroxydehydrogenase activity in the adult rat kidney. Identification, assay, pathway, and some enzyme properties.

Prostaglandin F2alpha is converted to 15-keto-13,14-dihydroprostaglandin E2 by adult rat kidney homogenates. A variety of substrates labeled as either the 9beta position alone or at several other positions in the prostaglandin molecule were used to define the step at which the crossover from the F type to the E type prostaglandins takes place. Time course studies further confirmed that 15-keto-13,14-dihydroprostaglandin F2alpha is the immediate substrate for this enzyme which we have termed prostaglandin 9-hydroxydehydrogenase. An assay system based on specific loss of tritium from 9beta-tritiated prostaglandin F2alpha is described. Enzyme activity with prostaglandin F2alpha as substrate is linear with time up to 10 min, stimulated by NAD+, saturable at low concentrations of substrate, stable to storage at minus 25 degrees in phosphate buffer (up to 3 weeks), and has a broad pH optimum around 7.5. The product, 15-keto,13,14-dihydroprostaglandin E2 was identified by mass spectrometry through a sodium borohydride-sodium borodeuteride reduction method.


Kinetics of the oxidation of p-aminobenzoic acid catalyzed by horseradish peroxidase compounds I and II.

The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.


Properties of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductase from pig kidney. Amino acid composition, reactivity of cysteinyl residues, and stereochemistry of D-glyceraldehyde reduction.

Some physical and chemical properties of the monomeric NADP+-dependent aldehyde reductase (previously called TPN-L-hexonate dehydrogenase or D-glucuronate reductase) from pig kidney have been examined. The amino acid composition has been determined. Four of the five thiol groups react with p-mercuribenzoate at pH 7, with no resulting loss of catalytic activity. High concentrations of p-mercuribenzoate cause complete enzyme inhibition, which can be partly reversed by addition of aldehyde reductase is low (9%, estimated from the ellipticity at 208 nm), and 70 to 80% of the tyrosine and tryptophan residues aare buried within the molecule. One molecule of NADPH binds to the enzyme (Kp equal 25 muM), causing a blue shift and enhancement of the coenzyme fluorescence, and suggesting that the environment of the active site is hydrophobic. In the reduction of D-glyceraldehyde, catalyzed by aldehyde reductase, the pro-4R "A" hydrogen of NADPH attacks the re face of the carbonyl group. This stereospecificity is the same as in the reductions of D-glyceraldehyde and acetaldehyde effected by rabbit muscle dehydrogenase and liver alcohol dehydrogenase, respectively.


Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.


Some physical and kinetic properties of adenylyl sulfate reductase from Desulfovibrio vulgaris.

Adenylyl sulfate reductase has been purified from the anaerobic sulfate-reducing bacterium, Desulfovibrio vulgaris, and judged to be homogenous by several criteria. Different forms of the enzyme could be visualized in polyacrylamide gels after electrophoresis and these polymeric species have been studied by a combination of absorption spectra, polyacrylamide gel electrophoresis, and sedimentation  velocity experiments. A dimeric species of molecular weight 440,000 is stable in potassium phosphate buffer but can be dissociated to a 220,000 molecular weight species by either changing the buffer system to Tris-maleate or addition of AMP, DAMP, or adenylyl sulfate. Other catalytically active nucleotides are not capable of effecting this dissociation. The enzyme was determined to contain 12 non-heme irons, 12 acid-labile sulfides, and 1 FAD per molecule when calculated on the basis of a monomeric molecular weight of 220,000. ;el electrophoresis in the presence of sodium dodecyl sulfate indicated subunits of molecular weight 72,000 and 20,000. The extinction coefficient when determined in phosphate buffer at 372 nm is 108,000 M-1 cm-a. Steady state kinetic experiments employing ferricyanide, cytochrome c, and reduced methyl viologen as artificial electron transfer reagents were performed and the kinetic constants obtained under various conditions. Several nucleotide substrates were employed and compared in each assay with respect to Km and Vmax. The reduction of cytochrome c was found to be sensitive to both anaerobiosis and superoxide dismutase, suggesting the involvement of superoxide anions with this electron acceptor.


Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.


A protein from Ustilago which forms an acid-soluble complex with deoxyribonucleic acid.

A protein which can render DNA largely acid-soluble has been purified 1600-fold from high salt extracts of Ustilago maydis. The activity is unusual in that substrate DNA is not made acid-soluble through hydrolysis to small oligomers. Rather, the protein appears to bind to DNA to form a complex which itself is acid-soluble. The activity of conversion of DNA to an acid-soluble form is cold-labile, but the inactivation by cold is reversible by brief heat treatment. Divalent cations stimulate the activity; phosphate is inhibitory. Optimal activity is observed at pH 6.0 and again at pH 9.0. Nucleoside triphosphates and diphosphates stimulate activity at low protein concentrations but are not hydrolyzed during the course of reaction. The protein behaves anomalously on gel filtration columns and is completely excluded by Sephadex or agarose gels. When analyzed by sedimentation velocity, the protein was found to sediment at 5.3 S, the same rate at which a globular protein of 65,000 daltons would sediment. Dependence of activity upon protein concentrations is sigmoidal. K+ and to a lesser degree NH4+ are partially effective in abolishing the lag in the concentration curve. The protein displays a saturation curve when exposed to increasing DNA concentrations. Such a curve could only result from a non-random or cooperative mode of binding of the protein to DNA. A mutant sensitive to gamma and ultraviolet radiation with an abnormally high level of the protein has been found. Haploid populations of the mutant grow slowly and contain a large proportion (10 to 20%) of inviable cells. Diploids are defective in mitotic allelic recombination and fail to complete meiosis. It is speculated that the protein may be important in the regulation of chromosome condensation.


Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.

The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.


Purification and regulation of fructose-1,6-bisphosphatase from Bacillus licheniformis.

The fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the spore-forming bacterium Bacillus licheniformis was purified approximately 800-fold (with a 20% yield of activity) by a procedure that included ammonium sulfate precipitation, precipitation by MnCl2, and gamma-alumina gel absorption. Catalysis by this enzyme in vitro was specific for fructose 1,6-bisphosphate (Km of approximately 20 muM) and proceeded optimally at pH 8.0 to 8.5. Fructose-1,6-bisphosphatase was found to be rapidly inactivated by incubation in the presence of AMP or in the absence of Mn2+. The AMP inactivation was prevented by adding P-enolpyruvate to the incubation mixture. The enzyme was slowly inactivated when incubated in the presence of stabilizing concentrations of Mn2+ (5 mM) at protein concentrations of less than 8 mg of protein per ml. An additional system is produced during sporulation which specifically inactivates fructose bisphosphatase in vitro. This system, which is distinctly different from the AMP inactivating system, can be blocked by P-enolpyruvate. This fructose bisphosphatase, like fructose bisphosphatases from other sources, was strongly inhibited by AMP, exhibiting a Ki of approximately 5 muM. This inhibition, however, could be completely overcome by P-enolpyruvate. P-enolpyruvate was also found to be an activator of the enzyme and exhibited a Km of approximately 2 muM. This activation was prevented in a competitive manner by AMP, exhibiting a Ki of approximately 5 muM. No other effector of fructose bisphosphatase was identified in an extensive search. The specific activity of fructose bisphosphatase in crude extracts was found to be independent of the stage of the life cycle of the bacterium or of the nature of the carbon-energy source supporting growth. Immunoprecipitation studies indicate that no new species of fructose biphosphatase is produced during gluconeogenic growth or sporulation. The enzyme extracted from cells under a variety of physiological conditions exhibited a molecular weight of about 5 times 10-5 as determined by sucrose density centrifugation. Therefore, it is proposed that a single constitutively synthesized fructose bisphosphatase is present in B. licheniformis. Measurements of the intracellular level of fructose 1,6-bisphosphate indicate that the variation in the level of substrate throughout growth (1 mM) and sporulation (0.3 mM) does not regulate the in vivo activity of this enzyme, since the Km of the enzyme for fructose 1,6-bisphosphate is approximately 10-fold lower than the lowest in vivo concentration of substrate. P-enolpyruvate is proposed as the major regulator of fructose bisphosphatase activity in vivo.


Dissociation of human follicle-stimulating hormone. Comparison with luteinizing hormone.

Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.


The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.


Characterization of the different polypeptide components and analysis of subunit assembly in ferritin.

Ferritin was dissociated into subunits by various denaturants and the subunits were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Human, horse, rat, and rabbit ferritins all exhibited characteristic patterns of heterogeneity; components with molecular weights of about 19,000, 11,000, and 8,000 were invariably found in these preparations. This result contradicts earlier reports that ferritin consists of 24 identical subunits. These polypeptides were isolated, purified in the presence of low concentrations of detergent, and characterized. Evidence based on amino acid compositions, NH2-terminal analysis and investigation of detergent-induced breakdown products, indicated that the 19,000 molecular weight component is a composite of the 8,000 and 11,000 molecular weight chains. Circular dichroism studies showed that the 19,000 molecular weight polypeptide retained appreciable amounts of ordered secondary structure whereas the two lower molecular weight peptides were unfolded to a much greater extent. If the 8,000 and 11,000 molecular weight polypeptides were recombined in equimolar amounts and the denaturant was completely removed, a substance with electrophoretic mobility and morphological appearance of native apoferritin was obtained.


The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.


Regulation of dopamine beta-hydroxylase in rat adrenal glands.

Rat adrenal gland levels of dopamine beta-hydroxylase are subject to dual control. Activation of the splanchnic nerves to the adrenal medulla by reserpine induces the synthesis of dopamine beta-hydroxylase without altering the rate of enzyme degradation. In contrast, hypophysectomy causes a decline in steady state dopamine beta-hydroxylase levels by first accelerating the rate of degradation, then by slowing the rate of enzyme synthesis as well. Adrenocorticotropic hormone administration partially reversed the effect of hypophysectomy on dopamin beta-hydroxylase degradation. These findings suggest that the trans-synaptic factors controlling dopamine beta-hydroxylase induction act by a different mechanism (enzyme synthesis) than the hormonal controls regulating steady state levels (enzyme degradation). Thus, active inhibition of enzyme degradation may be an important control in maintenance of steady state enzyme levels.


Glycoprotein biosynthesis: studies on thyroid mannosyltransferases. I. Action on glycopeptides and simple glycosides.

A particulate fraction from calf thyroid catalyzes the transfer of mannose from GDP-mannose to exogenous glycopeptides and methyl or aryl glycosides to form alpha-D-mannopyranosyl-D-mannose sequences. The transfer to the simple glycosides required a single nonreducing mannose residue linked to a lipophilic aglycone. Thus p-nitrophenyl-, 4-methylumbelliferyl-, phenyl- and methyl-alpha-D-mannopyranosides were effective acceptors while free mannose and glycosides of several other sugars were totally inactive. The Km value for methyl-alpha-D-mannopyranoside was 2.6 mM. Specificity for the anomeric configuration of the acceptor was glycosylated to the extent of 50% of the alpha anomer and mutual inhibition between these two acceptors was observed. Acetolysis or mild acid hydrolysis of the 14C-labeled products from the glycoside acceptors yielded the disaccharide, 2-O-alpha-D-mannopyranosyl-D-mannose, which represents the predominant linkage between mannose residues in the carbohydrate unit A of thyroglobulin. Glycopeptides with mannose sequences served as acceptors for the transfer reaction but only after dinitrophenylation of their peptide portion. The unit A glycopeptides of thyroglobulin with 10 mannose residues (Km equals 0.89 mM) were much better acceptors than glycopeptides containing the core portion of unit B which contains only three mannose components. Reduction in size of unit A glycopeptide acceptors by timed alpha-mannosidase treatment resulted in a progressive decrease in activity. Peptide-free unit A was inactive even after it was modified to carry dinitrophenyl groups on its glucosamine residues. GDP-mannose was the most effective glycosyl donor, with a Km value of 1.4 muM for methyl-alpha-D-mannopyranoside and 0.30 muM for dinitrophenyl unit A glycopeptides, although  ADP- and UDP-mannose could substitute to the extent of 40 to 45%. The mannose transfer to the glycopeptides had a optimum of 6.3 while that to the simple glycopeptides was best at pH 7.0. Both types of transfer reactions required a divalent cation with manganese serving most effectively in that capacity. Mannoslytransferase activity for both groups of acceptors was found predominantly in particulate subcellular fractions. A number of aromatic compounds and reagents which are disruptive of membrane integrity caused loss of enzyme activity presumably by interfering with the function of the lipophilic substituents on the various acceptors.


Biochemical aspects of cardiac muscle differentiation. Deoxyribonucleic acid synthesis and nuclear and cytoplasmic deoxyribonucleic acid polymerase activity.

DNA synthesis and DNA polymerase activity have been measured in terminally differentiating cardiac muscle of the rat. Incorporation of [3H]thymidine into DNA essentially ceases by the 17th day of postnatal development. Cardiac muscle of neonatal rats contains at least two molecular species of DNA polymerase: a 3.5 S DNA polymerase that can be extracted from nuclei with 0.2 m potassium phosphate and a 6 to 8 S soluble cytoplasmic DNA polymerase. The nuclear DNA polymerase in crude extracts has a pH optimum of 9.0 and is more active with native DNA than with denatured DNA as the primer-template. The cytoplasmic DNA polymerase in crude extracts has a pH optimum of 7.5 and is more active with denatured DNA. The activity of the 6 to 8 S cytoplasmic DNA polymerase decreases 80-fold from day 1 to day 17 after birth, which correlates temporally with the reduced rate of DNA synthesis. The activity of the 3.5 S nuclear DNA polymerase remains relatively constant throughout postnatal development. Mixing experiments (assay of neonatal enzyme extracts with adult enzyme extracts) gave additive results, suggesting that the decline in 6 to 8 S DNA polymerase activity apparently is not due to the presence of absence of soluble activators or inhibitors at different times during development. These studies may provide a system which can be used to investigate the control of DNA synthesis and cellular proliferation during the terminal stages of cardiac muscle differentiation.


Enhancement of the glutaminase activity of carbamyl phosphate synthetase by alterations in the interaction between the heavy and light subunits.

Glutamine-dependent carbamyl phosphate synthetase (from Escherichia coli) was previously shown to be composed of a light subunit (molecular weight similar to 42,000) which has the binding site for glutamine and a heavy subunit (molecular weight similar to 130,000) which has binding sites for the other reactants and allosteric effectors. The subunits may be separated with retention of catalytic activities; only the separated light subunit exhibits glutaminase activity. The previous finding that storage of the native enzyme at pH 9 at 0 degrees increased its glutaminase activity by about 25-fold was further investigated; such storage markedly decreased the glutamine- and ammonia-dependent synthetase activities of the enzyme. Treatment of the enzyme with p-hydroxymercuribenzoate led to transient increase of glutaminase activity followed by inhibition. When the enzyme was treated with N-ethylmaleimide or with 5,5'-dithiobis-(2-nitrobenzoate), the glutaminase activity was increased by about 250-fold with concomitant loss of synthetase activities. The enhancement of glutaminase produced by storage of the enzyme at pH 9 was associated with intermolecular disulfide bond formation and aggregation of the enzyme. Aggregation also was observed after extensive treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) or N-ethylmaleimide. However, a moderate increase of glutaminase activity (about 30-fold) was observed without aggregation under conditions in which one sulfhydryl group on the light subunit reacted with either reagent. The findings suggest that the increased glutaminase activities observed here are associated with structural changes in the enzyme in which the intersubunit relationship is altered so as to uncouple the catalytic functions of the enzyme and to facilitate access of water to the glutamine binding site on the light subunit.


Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.