Biomedical subjects

M J Kornblatt

Publications and source records attributed to M J Kornblatt.

At least 19 recordsLinked to original sources

PubMed1998

Can monomers of yeast enolase have enzymatic activity?

Using a combination of ultraviolet spectroscopy under pressure and stopped-flow kinetics under pressure, we have shown that the monomers of yeast enolase produced by hydrostatic pressure are inactive. K(eq), deltaV and deltaV for the dissociation/inactivation produced by hydrostatic pressure have been determined under various conditions. Removing the Mg2+ from enolase, either by adding EDTA or by preparing apoenzyme, displaces the equilibrium towards monomers and decreases both deltaV and deltaV. Loss of Mg2+ contributes to the negative deltaV for dissociation; this loss occurs, at least partially, in the transition state for dissociation. Both removal of Mg(II) and dissociation of the enzyme produce major changes in the intensity of the aromatic region of the CD spectrum. We propose that these changes in the CD spectra reflect changes in the conformations of the 'mobile loops' of enolase. The precise conformation of these, loops is necessary for binding Mg2+ (and, hence, for activity) and for maintaining subunit interactions.

Circular Dichroism↗

PubMed1998

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.

Animals↗

PubMed1996

Mechanism of rabbit muscle enolase: identification of the rate-limiting steps and the site of Li+ inhibition.

Steady-state and non-steady-state techniques have been used to identify the rate-limiting steps for beta beta enolase (rabbit muscle enolase), at pH 7.1, with Mn2+ as the required cation. A minimum mechanism for enolase includes eight steps, [see text] where S is phosphoglycerate, P is phosphoenolpyruvate (PEP), I is the carbanion intermediate, M is Me2+ and EM is the holoenolase (i.e., the first Me2+ is bound). Asterisks represent a different conformation of the quaternary complexes. At pH 7.1, the primary kinetic isotope effect = 1, and kappa(cat) decreases as solvent viscosity increases. The changes in protein fluorescence that occur upon substrate binding and product release [EMSM <-> (EMSM)* and (EMPM)* <-> EMPM] were followed by stopped-flow fluorimetry; the viscosity dependence of the observed rates was also determined. The data support the following mechanism. Product formation is fast and precedes the slow steps of the reaction, consistent with the observation of a pre-steady-state burst of PEP. The rate-limiting steps are kappa(+6) the conformational change associated with product release, and kappa(+8) the dissociation of PEP. Li+ inhibits the activity of enolase by increasing kappa(+6) and kappa(-3), thus decreasing the steady-state concentration of (EMSM)*.

Animals↗

PubMed1996

The effects of sodium perchlorate on rabbit muscle enolase--Spectral characterization of the monomer.

Incubation of rabbit beta beta enolase in NaClO4 (< or = O.3 M) results in a loss of enzymatic activity and striking changes in the second-derivative ultraviolet spectrum of enolase. HPLC gel filtration shows that dissociation of the dimeric enzyme is occurring. We have used molecular modelling, fluorescence and circular dichroic spectroscopy to examine the structural differences between the monomeric and dimeric forms of this protein. In the dimer, the tyrosine residues are in a non-polar environment; upon dissociation, two of them that were at the dimer interface become exposed. This results in large changes in the second-derivative spectrum. Both the tryptophan fluorescence emission spectrum and the aromatic region of the CD spectrum indicate that there are also changes in the environment of other aromatic residues. No perturbations in the peptide bond region of the CD spectrum are observed. We propose that the major structural effect of NaClO4 is to increase the flexibility of the loops connecting the helices and strands of the alpha/beta barrel of enolase. These loops, which contain about half of the aromatic residues, contain some of the residues of the active site and other residues involved in subunit contacts. Increased flexibility of the loops could disrupt both subunit interactions and the structure of the active site.

Amino Acids↗

PubMed1995

Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions.

Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast enolase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers dissociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyrosines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges and hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrostatic Pressure↗

PubMed1994

Warm brown fat.

Explore the source record for details and available documents.

Adipose Tissue, Brown↗

PubMed1993

The role of water in the dissociation of enolase, a dimeric enzyme.

Exposure of enolase to hydrostatic pressure results in a reversible inactivation of the enzyme; increasing the osmotic pressure, by adding glycerol, glucose, or sucrose to the solutions stabilizes the enzyme against the effects of hydrostatic pressure. The effects of both hydrostatic and osmotic pressure on the rate of inactivation have been determined. As hydrostatic pressure increases, the rate of inactivation increases. As osmotic pressure increases, the rate of inactivation decreases. We have interpreted these results using the following model: hydrostatic pressure causes the active, dimeric enzyme to dissociate into inactive monomers; during the dissociation, the subunit interfaces become hydrated. As osmotic pressure increases, hydration becomes more difficult and dissociation is reduced. The combined effects of hydrostatic and osmotic pressure suggest that much of this hydration occurs during formation of the transition state.

Animals↗

PubMed1993

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.

Animals↗

PubMed1992

Characterization of an endopeptidase of Trypanosoma brucei brucei.

A soluble 80-kDa endopeptidase has been isolated from Trypanosoma brucei brucei. The enzyme, which has a pI 5.1, is optimally active at about pH 8.2 and has apparent pKa values of 6.0 and greater than or equal to 10. It is inhibited by the serine protease inhibitor diisopropylfluorophosphate and by the serine protease mechanism-based inhibitor 3,4-dichloroisocoumarin. Unexpectedly, the enzyme is inhibited by the cysteine protease inhibitor benzyloxycarbonyl-Leu-Lys-CHN2 but not by the related diazomethane, butoxycarbonyl-Val-Leu-Gly-Lys-CHN2, nor by other cysteine protease specific compounds. Specificity studies with a variety of amidomethylcoumaryl (AMC) derivatives of small peptides show that the enzyme has a highly restricted trypsin-like specificity. The best substrate, based on the magnitude of kcat/Km, was benzyloxycarbonyl-Arg-Arg-AMC; other good substrates were benzyloxycarbonyl-Phe-Arg-AMC, benzoyl-Arg-AMC, and compounds with Arg at P1 and Ala or Gly at P2. The hydrolysis of most substrates obeyed classical Michaelis-Menton kinetics but several exhibited pronounced substrate inhibition. The enzyme did not activate plasminogen nor decrease blood clotting time; it was inhibited by aprotinin but not by chicken ovomucoid. We conclude that the enzyme is a trypsin-like serine endopeptidase with unusually restricted subsite specificities.

Amino Acid Sequence↗

PubMed1990

The inhibition of yeast enolase by Li+ and Na+1.

The activity of yeast enolase is inhibited by Li+ and Na+. At pH 7.1, inhibition by Li+ is "mixed" with respect to Mg2+; both Vmax and Km (Mg2+) are increased by Li+. The inhibition by Li+ appears to be partial, indicating that enzyme with Li+ bound is active. The step inhibited by Li+ cannot be proton abstraction since Li+ decreases the kinetic isotope effect on Vmax. At pH 9.2, where proton abstraction is no longer partially rate-limiting, inhibiton by Li+ is competitive with respect to Mg2+. The rate of enzyme-catalyzed exchange of the C-2 hydrogen with solvent is not affected by Li+. We interpret these results as follows: Li+ (and Na+) binds to enolase and decreases the rate of at least one step in the mechanism. At pH 7.1, this step is partially rate-limiting; at pH 9.2, this step is a fast step in the reaction. The step inhibited by Li+ cannot be proton abstraction but may be release of product (phosphoenol pyruvate) or Mg2+.

Hydrogen-Ion Concentration↗

PubMed1990

The salt-induced dissociation and inactivation of a mammalian enolase: evidence for the formation of active monomers.

The gamma gamma isozyme of rabbit enolase was labeled with fluorescein and the effects of NaClO4 on both enzymatic activity and fluorescence polarization were studied. NaClO4, but not NaCl, dissociates and partially inactivates the enzyme. If dissociation is prevented, either by the addition of substrate or by covalently crosslinking the enzyme, inactivation is also prevented. Analysis of the time and concentration dependence of inactivation and dissociation shows that the decrease in activity is a two-step process: D in equilibrium 2M in equilibrium 2M*. Both monomeric forms of the enzyme are catalytically active.

Animals↗

PubMed1987

The pressure-induced inactivation of mammalian enolases is accompanied by dissociation of the dimeric enzyme.

The effects of exposure to pressure on both the activity and the quaternary structure of rabbit brain enolases, forms alpha alpha, alpha gamma, and gamma gamma were studied in the pressure range of 1 to 3400 bar. Effects on quaternary structure were determined by subunit scrambling (the formation of alpha alpha and gamma gamma from alpha gamma or vice versa). All three dimers are stable up to pressures of 1200 bar. The dissociation of gamma gamma begins at 1200 bar, yielding a stable monomer; inactivation of gamma gamma does not begin until the pressure is greater than 2000 bar. Dissociation of gamma gamma is not accompanied by changes in the tryptophan fluorescence of the protein. However, the fluorescence does decrease when the pressure is greater than 2000 bar, the point at which inactivation of gamma gamma starts. The alpha monomer, on the other hand, is unstable in the pressure range that produces dissociation of alpha alpha. This process, which also begins at 1200 bar, is paralleled by inactivation. Crosslinking the enzyme with glutaraldehyde demonstrated that the inactive form of the enzyme is monomeric. The pressure-induced inactivation of these forms of enolase is thus clearly a two-step process, with both dissociation and inactivation occurring. The difference in pressure sensitivity of rabbit brain alpha alpha and gamma gamma is due to a difference in stability of the alpha and gamma monomers and not due to a difference in the pressures required for dissociation.

Animals↗

PubMed1986

Inhibition of yeast cytosine deaminase by 5-bromo-2-pyrimidinone and its covalent hydrate.

Yeast cytosine deaminase (EC 3.5.4.1) is inhibited by 5-bromo-2-pyrimidinone. In aqueous solution at neutral pH three forms of this compound (the anion, the parent, and the covalent hydrate) are in equilibrium. Experiments were undertaken in order to determine the relative contributions of these three forms to the observed inhibition. The anion makes little or no contribution. Both the parent and the covalent hydrate inhibit the enzyme, with the Ki for the hydrate being 0.2-0.02 times that of the parent. In the presence of stoichiometric concentrations of the enzyme, the equilibrium between parent and hydrate is displaced towards the hydrate; however, the hydration is not catalyzed by cytosine deaminase.

Cytosine Deaminase↗

PubMed1983

Characterization and localization of alkaline phosphatase activity in rat testes.

Alkaline phosphatase activity in extracts of testes of sexually immature (13 days old) and sexually mature rats has been characterized by its heat sensitivity, the extent of inhibition by homoarginine and phenylalanine, and by polyacrylamide gel electrophoresis. The testicular enzyme appears to be a liver-bone-kidney-type alkaline phosphatase. There are no significant differences in the properties of the enzyme from animals of these two ages. Spermatocytes and early spermatids contain very little alkaline phosphatase activity; the specific activity of a nonflagellate germinal cell suspension is only 1/20th that of the whole testis. Since the constant level of activity in immature and mature animals is not consistent with the enzyme activity being present only in late spermatids, we conclude that the majority of the testicular enzyme is present in nongerminal cells. The presence of alkaline phosphatase in plasma membrane purified from testes of adult rats was demonstrated.

Alkaline Phosphatase↗

PubMed1983

Changes in the expression of the alpha alpha form of enolase during neuroblastoma differentiation.

The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form alpha alpha, and an increase in the activity associated with the gamma-containing isozymes (alpha gamma plus gamma gamma); in the absence of DMSO, there is no decrease in alpha alpha or in total enolase activity. In order to study the mechanism of the changes in alpha alpha, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the alpha antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the alpha antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the alpha antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t1/2 greater than 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the alpha subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the alpha gene that occurs in vivo during neuronal differentiation.

Animals↗

PubMed1980

The subcellular localization of testicular sulfogalactoglycerolipid.

The subcellular localization of sulfogalactoglycerolipid in rat testicular germinal cells was determined. The sulfolipid of young rats was labelled in vivo with Na235SO4. Rat testis cell suspensions were prepared, homogenized, and centrifuged on linear, continuous, sucrose gradients. The labelled lipid had the identical equilibrium density distribution pattern as alkaline phosphatase, an enzyme of the plasma membrane. The pattern of the sulfolipid was different from the patterns of enzyme markers for the Golgi apparatus, lysosomes, mitochondria, and endoplasmic reticulum. From these results, we conclude that sulfogalactoglycerolipid is located on the plasma membrane of rat testicular germinal cells.

Animals↗

PubMed1979

Synthesis and turnover of sulfogalactoglycerolipid, a membrane lipid, during spermatogenesis.

The synthesis and turnover of sulfogalactoglycerolipid (SGG) were studied by in vivo labelling of SGG with 35S. The loss of [35S]SGG from the testes and its appearance in the vas deferens plus epididymis were followed with time. DNA was labelled by administration of [3H]thymidine and the behavior of the two isotopes was compared. The results demonstrate that SGG snythesis occurs only in very early spermatocytes and that, once made, the compound does not turn over. The SGG is lost from the testes when germinal cells die or mature into spermatozoa.

Animals↗

PubMed1975

Isolation and partial characterization of a sulfogalactoglycerolipid from rat brain.

The brain of adult rats were analyzed for the presence of 35-SO4-containing glycolipids following intraventricular injection of Na2-35SO4. Radiochromatographic analyses revealed the presence of two minor 35-SO4-containing glycolipids, in addition to sulfogalactosylceramide. One of these two minor sulfolipids was isolated and tentatively identified as a 1-O--alkyl-2-0-acyl-3-(3'-sulfogalactosyl)-glycerol, a compound recently demonstrated to be the major glycolipid of mammalian testis. The alkyl and acyl compositions of the compound from rat brain are more heterogeneous than those from rat testis. The non-sulfated form of the galactoglycerolipid was also detected in rat brain. The amount of the sulfogalactoglycerolipid in rat brain is 0.19 mumol per gram wet weight, approximately one-third of the amount in rat testis (per gram wet weight), and is approximately one-fifteenth that of sulfogalactosylceramide in rat brain. The possible significance of the common occurrence in brain and testis of sulfated and non-sulfated galactolipids is discussed.

Animals↗