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F Jordan

Publications and source records attributed to F Jordan.

At least 73 records · Page 4Linked to original sources
PubMed1989

Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process.

Subtilisin E, an alkaline serine protease consisting of a single polypeptide chain of 275 amino acids is produced from a pre-pro-protein. The pre-sequence functions as the signal peptide for protein secretion across the membrane. Deletion of the pro-sequence yields mature but inactive subtilisin: the 77-amino acid pro-sequence must precede the mature subtilisin to guide the latter into an active conformation. Pro-subtilisin denatured in 6 M guanidine-HCl can be self-processed to the active enzyme intramolecularly, with concomitant cleavage of the pro-sequence, when dialysed against renaturing buffer. We have constructed an active-centre mutant of pro-subtilisin (Asp 32----Asn) which is not processed to active enzyme, unlike the wild-type pro-subtilisin, because intramolecular processing is prevented. Here we report an intermolecular pathway for the refolding of the inactive mature protein to an active enzyme in vitro with the aid of exogenously added pro-sequence. We establish conditions under which the mature inactive form, as well as acid-denatured subtilisins Carlsberg and BPN', can be renatured by the mutant pro-subtilisin.

Enzyme Activation↗
PubMed1989

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region.

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenodoxin↗
PubMed1989

Multinuclear magnetic resonance studies on serine protease transition state analogues.

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.

Boron Compounds↗
PubMed1989

Regional in vivo superfusion of the spinal cord and KC1-induced amino acid release.

In vivo push-pull superfusion was used to sample the regional release of amino acids into spinal superfusates of urethane-anesthetized rats. By collecting superfusates from the intrathecal space surrounding the sacral-lower thoracic spinal cord, it was possible to achieve a stable release of amino acids in one- and five-minute superfusate fractions. Introducing the depolarizing agent, potassium chloride (KC1) (40 mM), into the superfusion medium significantly increased GLU, GLY, and TAU concentrations in superfusates compared to pre-KCl values. The findings that these three amino acids were the only ones (out of 20) that showed a significant increase in response to KCl administration, suggest that they mediate neurotransmission in this region of the spinal cord. Amino acid concentrations were determined in spinal superfusates by high performance liquid chromatography (HPLC), utilizing an automated ortho-phthaldialdehyde precolumn derivatization system. The regional superfusion system described in this paper provides a technique for measuring KC1-produced release of neurochemicals that may mediate neurotransmission in delimited spinal regions.

Amino Acids↗
PubMed1989

Hypersensitivity to carbon dioxide in panic disorder.

Seven male panic patients did not panic but were significantly more sensitive to steady-state carbon dioxide inhalation than five male normal control subjects. The male patients' hypersensitivity to carbon dioxide was unrelated to current state of anxiety or acute panic.

Anxiety↗
PubMed1988

Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin.

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.

Carbon Isotopes↗
PubMed1988

Characteristics of fatal gunshot wounds in the home in Oklahoma: 1982-1983.

Children are a unique group at risk of injury from firearms because of their immaturity, curiosity, and imitative behavior. All unintentional firearm deaths in persons younger than age 20 years that occurred in Oklahoma in 1982 and 1983 were reviewed. There were 32 unintentional deaths from firearms in children from birth to age 19 years. The death rate in rural counties was four times that of urban counties. Twenty-seven deaths (85%) occurred at home, with an adult present in only two cases. The home death rate for males was 5.2 times that of females, with 15- to 19-year-old males most at risk. The rates among whites and Native Americans were similar, at 1.5 and 1.2 per 100,000, respectively, with no deaths among the black population. This review concurs with previous studies that firearms are a significant cause of mortality in the pediatric age group. Counseling parents about the hazards of firearms may prevent deaths through better supervision and more responsible gun care and storage.

Accidents, Home↗
PubMed1988

Stimulation by mammalian insulin of glycogen metabolism in a wall-less strain of Neurospora crassa.

Addition of bovine insulin to cells of the wall-less variant FGSC4761 of Neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17-18% (P less than 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P less than 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6-phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells.

Dose-Response Relationship, Drug↗
PubMed1988

Studies on the biosynthesis of the antibiotic crisamicin A and carbon-13 magnetic resonance assignments.

The biosynthesis of crisamicin A, a novel dimeric isochromanequinone antibiotic from Micromonospora purpureochromogenes subsp. halotolerans has been investigated by [1-13C] and [2-13C] labeled acetate precursor feeding experiments. Analysis of the proton noise decoupled and off resonance 13C NMR spectra of 13C enriched and unenriched crisamicin A and their acetate derivatives indicated the biosynthesis via the polyketide pathway, as expected. Further analysis of the enriched spectra allowed the complete assignment of the carbon signals. Of particular interest was the establishment of the linkage between the two monomeric halves of the molecule and determination of the location of the phenolic hydroxyls.

Anti-Bacterial Agents↗
PubMed1988

Rectus sheath hematomas: their pathogenesis, diagnosis, and management.

Rectus sheath hematomas are an uncommon entity causing an abdominal pain that may mimic other serious intraabdominal conditions. Knowledge of its predisposing factors and presentation are paramount in making the diagnosis. To facilitate the understanding of this condition, the authors reviewed their experience at St. Joseph Mercy Hospital in Ann Arbor, Michigan for the period between 1977 and 1986. Eight patients who were hospitalized for other reasons were found to have rectus sheath hematomas. All patients were women with an age range of 26 to 85 years and a mean age of 58 years. The correct diagnosis was made clinically in four patients. In those in whom hematomas of the rectus sheath were not initially diagnosed, surgery was undertaken, at which time the condition was identified. Predisposing factors include trauma, childbirth, previous operation of the lower abdomen, and anticoagulant use. Ultrasonography may aid in the diagnosis but CT scan is most accurate in its ability to define the lesion. When diagnosed clinically, a conservative therapeutic program can usually be instituted.

Abdominal Muscles↗
PubMed1986

Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

Carboxy-Lyases↗
PubMed1986

Conjugated alpha-keto acids as mechanism-based inactivators of brewer's yeast pyruvate decarboxylase: electronic effects of substituents and detection of a long-lived intermediate.

A series of phenyl substituted E-4-phenyl-2-keto-3-butenoic acid derivatives were synthesized (p-Cl, m-Cl, p-NO2, m-NO2, o-NO2, 3,4-Cl2, 2,6-Cl2, p-CH3O, p-(CH3)2N) and tested as potential irreversible inhibitors of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1). All those derivatives with electron withdrawing substituents were found to be time-dependent inactivators of the enzyme, unlike the p-CH3O- and p-(CH3)2N derivatives. Detailed kinetic studies with the m-nitro derivative (the most potent inhibitor) indicated that this compound formed reversible complexes with the enzyme at two sites (supposed regulatory and catalytic with Ki values of 0.026 and 0.13 mM, respectively) prior to irreversible inactivation of the enzyme. In addition, concurrently with the inactivation, addition of the m-NO2 derivative to the enzyme produced a new VIS absorbance with lambda max near 430 nm. This absorbance was attributed to the enzyme-bound enamine intermediate. The time course of formation and disappearance of the intermediate could be determined and provided detailed information about the mechanism of the enzyme.

Carboxy-Lyases↗
PubMed1985

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins.

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

Acetaldehyde↗
PubMed1983

Direct spectroscopic observation of a brewer's yeast pyruvate decarboxylase-bound enamine intermediate produced from a suicide substrate. Evidence for nonconcerted decarboxylation.

The conjugated alpha-keto acid (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid, a suicide substrate (Kuo, D. J., and Jordan, F. (1983) Biochemistry 22, 3735-3740), when reacted with brewer's yeast pyruvate decarboxylase (EC 4.1.1.1), was found to produce a new absorption band centered at 440 nm. The band was attributed to the formation of a thiamindiphosphate-bound intermediate produced upon decarboxylation and was not observed in the absence of either thiamindiphosphate or apoprotein. Simultaneously, with the appearance of the spectral band, the enzyme was inactivated irreversibly. The combined evidence suggested that the spectral band pertains to an enzyme-bound conjugated enamine and the results constitute the first direct observation of such an intermediate in any thiamindiphosphate requiring enzymatic reaction. It could be concluded that for the alpha-keto acid employed, CO2 loss and C-protonation of the enamine take place in a stepwise, rather than concerted, manner and that the steps culminating in CO2 loss are faster overall than the subsequent steps that lead to the release of the product. The observation of stoichiometric concentration of enzyme-bound enamine intermediate supports the previous suggestion that alpha-hydroxyethyl thiamindiphosphate is not the true intermediate, but rather the enamine is (Ullrich, J., and Mannshreck, A. (1967) Eur. J. Biochem 1, 110-116).

Amines↗