Spectroscopic properties of tetrapyrroles on denatured biliproteins

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Alteration of thermal stability of glucose oxidase associated with the redox states

By the method of differential scanning calorimetry, it was found that thermal stability of glucose oxidase was dependent on its redox states. The oxidized form showed an apparent denaturation temperature at 76°C and the denaturation enthalpy was approximately 865 kcal/mol. On reduction of the enzyme, the denaturation temperature increased by about 10°, but no significant change was seen in the denaturation enthalpy. The activation energies of the denaturation of the oxidized and the reduced enzymes were about 89 and 103 kcal/mol, respectively. These results may imply conformational changes in the catalytic turnover of this enzyme.     

Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Dynamic information from protein crystallography. An analysis of temperature factors from refinement of the hen egg-white lysozyme structure.

A preliminary analysis is presented of whether the isotropic temperature factors derived from refinement of the tetragonal crystal structure of hen egg-white lysozyme at 2 Å resolution can be interpreted in terms of molecular motion. If the contributions to the temperature factors from experimental errors, crystal disorder and imperfections of the molecular model are neglected, the apparent thermal motion is found to be compatible with the pair of molecules that have the strongest interactions in the crystal either moving as a rigid-body with libration about a common axis, or vibrating in an intramolecular mode with the principal amplitudes radial to this axis. There is only weak evidence for hingebending, i.e. that the two lobes of lysozyme vibrate so that their separation varies. Side-chains that are exposed to the solvent and some segments of the main polypeptide chain (including residues 70–73, 82–84, 101–103) have greater apparent thermal motion than the remainder of the molecule.Although this analysis at a single temperature cannot establish whether thermal motion or static disorder (including conformational variability) underlies the observed effects, it suggests that the accurate determination of temperature factors will be useful in detailed studies of the dynamic properties of macromolecules.     

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as though they were ~ M_r 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 m guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.     

Cell surface proteins of Drosophila. II. A comparison of embryonic and ecdysone-induced proteins

The cell-surface proteins of Drosophila embryos at gastrula and myoblast fusion stages were characterized by radioiodination and two-dimensional gel electrophoresis. Over 13% of the cell surface proteins detected in gastrula embryos were not found in myoblast fusion stage embryos or in Drosophila embryonic cell line EH34A3 cells. Nearly 18% of the cell-surface proteins detected in myoblast fusion stage embryos were evident only at that stage. Embryonic cell-surface proteins were compared with cell-surface proteins from untreated EH34A3 cells and EH34A3 cells treated with 20-hydroxyecdysone, which induces cell aggregation and the expression of "new" proteins at the cell surface (D. F. Woods and C. A. Poodry, 1983, Dev. Biol. 96, 23-31). Only one of the proteins induced by ecdysone in EH34A3 cells was detected in the NP-40 soluble fraction of radioiodinated cell lysates, even after fractionation by lectin affinity chromatography and immunoprecipitation to enrich for putative ecdysone induced proteins. However, extraction of the NP-40 insoluble pellet of embryo cells revealed one additional protein that was present both in myoblast fusion stage embryos and hormone-treated culture cells. It was concluded that except for these two proteins, the cell-surface proteins induced in cultured cell lines by treatment with 20-hydroxyecdysone are not present in significant amounts in gastrula or myoblast fusion stage embryos.     

Purification and serological comparison of the yeast hexokinases P-I and P-II

An improved procedure for purification of the hexokinases P-I and P-II from baker's yeast is described. Yields, reproducibility, and purity are improved over those found by the methods used previously in this laboratory. The growth of large crystals of form P-I is described.Antisera prepared against the two purified hexokinases show only slight cross reaction by microcomplement fixation. The anti-sera have been used to demonstrate the presence of both P-I and P-II in crude extracts of various yeasts, including two haploid strains, and their absence in a yeast which contains glucokinase but no hexokinases.     

Characterization of ligand-induced states of maize homoserine dehydrogenase

The threonine-sensitive homoserine dehydrogenase (L-homoserine: NAD(P)+ oxido-reductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states (S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449-463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reacting-enzyme conditions provides direct support for the slow formation of a common steady-state species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD+, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.     

Hemoglobin Constant Spring: hemoglobin synthesis in heterozygous and homozygous states.

Thirteen adult and one newborn heterozygotes, and three homozygotes for hemoglobin Constant Spring were examined for globin chain synthesis. Reticulocytes from venous blood were incorporated with [³H]-leucine in an incubation mixture for 3 hours. Globin prepared from the radioactive, washed red cells was fractionated by CM-cellulose chromatography in 8 M urea and the total radioactivity of each globin chain was determined. The mean of $\text{α}\text{β}$ ratio in the heterozygotes was 1.34 ± SD 0.08, which is significantly different from that of 1.07 ± SD 0.03 in eleven normal controls. The $\text{α}\text{β+γ}$ ratio in the heterozygous neonate was also 1.39. The $\text{α}\text{β}$ ratios in the three homozygotes were around 1.6. The α-Constant Spring chain appears to be over produced, but it may be unstable or labile, not fully available for conjugation with the non alpha chains.     

The anal sac secretion of the red fox (Vulpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of the lion (Panthera leo)

Phenylacetic, 3-phenylpropionic, $\text{p}$-hydroxyphenylacetic and 3 ($\text{p}$-hydroxyphenyl) propionic acids together with the series of C₂ to C₆ saturated fatty acids previously reported in the anal sac secretion of the red fox ($\text{Vulpes vulpes}$) are identified as constituents of the anal sac secretion of the lion (Panthera leo). All these compounds are also observed in the anal sac secretion of the red fox using gas chromatography. The aerobic microflora of red fox and domestic dog ($\text{Canis familiaris}$) anal sac secretion samples invariably consisted predominantly of $\text{Streptococcus faecium}$ and $\text{Streptococcus faecalis}$. The hypothesis that the secretion volatiles so far identified may be microbiologically produced is examined.     

Ground and low lying electronic states of oxyhemoglobin from temperature dependent mōssbauer and susceptibility data

The temperature dependence of magnetic susceptibility and Mösbauer quadrupole splitting for oxyhemoglobin is considered to arise from contribution from a ground state singlet and two excited triplet electronic states. Using values of parameters obtained from fitting data for both measurements, the plausibility of this hypothesis is established.     

Interconversions between different sulfhydryl-related kinetic states in glucokinase

Rat liver glucokinase (EC 2.7.1.2) undergoes two distinct sulfhydryl-related reversible kinetic transitions. During normal assays in the presence of both substrates but without added reducing agents, the activity decays ("kappa" decay) over time to a new steady-state rate. The half-time for this decay is essentially constant at glucose levels from 2 to 200 mM and averages 6.2 +/- 2 min. Glucokinase in this kappa steady state displays an increased Km for glucose but has the same Vmax as normal, sulfhydryl-activated glucokinase. The kappa form does not itself exhibit kinetic cooperativity with glucose. In contrast, glucokinase incubated with neither glucose nor sulfhydryl reagents decays (mu decay) to a form whose Vmax is near zero. The t 1/2 for this transition is about 0.5 min at 0 or very low (0.5 mM) glucose concentrations. For both decays, incubations of enzyme with intermediate levels of reducing agents give steady-state mixtures of activated and either kappa and/or mu forms, depending on conditions during the decay. Enzyme at intermediate stages of the kappa decay displays an unchanged Vmax, intermediate (increased relative to activated enzyme) glucose S0.5 values, and diminished glucose cooperativity. In contrast, enzyme at intermediate steady-state mixtures of activated and mu forms has a normal glucose S0.5 and cooperativity but a diminished Vmax from the activated states. The enzyme at any stage of each decay may be fully reactivated by the addition of sulfhydryl reducing agents such as dithiothreitol, dithioerythritol, glutathione, or mercaptoethanol. A model is proposed to account for this complex behavior in glucokinase kinetics which proposes different enzymatic states (kappa and mu) locked in by sulfhydryl oxidation of different conformations dictated by glucose concentration. These sulfhydryl-related transitions may be important in regulation of glucokinase activity, since glucokinase is very sensitive (at least 20-fold differential activity) to concentrations of glutathione within the physiological range, perhaps allowing the normally variable glutathione levels or cytosolic redox potential to modify the rate of uptake and storage of blood glucose through control of glucokinase activity.     

A comparison of van't Hoff and calorimetric heats of binding to DNA using an ethidium selective electrode

A liquid membrane electrode selective for ethidium ion was used to measure free ethidium in mixtures with calf thymus DNA. Electrode response was unaffected by variation in ionic strength from 1 mm to 0.5 m, and was not degraded over the temperature range studied. DNA-ethidium binding isotherms obtained with the electrode at 17.4, 25.4, 30.1, and 40.6 °C were fitted to a single class of excluded sites model for $\text{}\text{?}$gn ranging from 0.01 to 0.16. van't Hoff analysis of these data yielded ΔH = ?8300 cal/mol ethidium bound (in 0.5 m KCl, 10 mm Tris buffer, pH 10, 1 mm EDTA). Direct calorimetric measurements of the heat of complex formation led to a value of ?7600 cal/mol at 25 °C in the same medium; the two results were not significantly different at the 95% confidence level. The agreement supports the validity of the ethidium selective electrode, and illustrates its utility in the study of ligand binding to nucleic acids and related materials.     

Acid-catalyzed esterification of lysozyme in methanol. Reactivities of individual carboxyl groups

Rats maintained on a diet low in phosphorus produce 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃ whether they have been thyroparathyroidectomized or not. On the other hand, rats maintained on low-calcium diets produce 1,25-dihydroxyvitamin D₃, but lose this ability within 48 hr after thyroparathyroidectomy. This loss of ability to synthesize 1,25-dihydroxyvitamin D₃ can be prevented or be restored by replacing their drinking water with calcium gluconate-glucose solution which returns their high serum inorganic phosphorus to normal levels. In thyroparathyroidectomized rats under a variety of conditions, the ability to synthesize 1,25-dihydroxyvitamin D₃ correlates with serum inorganic phosphorus values below 7–8 mg/100 ml while the ability to synthesize 24,25-dihydroxyvitamin D₃ correlates with serum phosphorus values above 7–8 mg/100 ml. There is in addition a close correlation between reduced kidney cortex inorganic phosphorus levels and the synthesis of 1,25-dihydroxyvitamin D₃. It is suggested that the renal tubular cell inorganic phosphorus level underlies the regulation of synthesis of 1,25-dihydroxyvitamin D₃ in the kidney and that the parathyroid hormone and calcitonin regulate 1,25-dihydroxyvitamin D₃ synthesis via their effects on renal cell inorganic phosphorus levels.     

A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides

The polysaccharide secreted by Klebsiella aerogenes type 54 strain A3 was isolated, methylated, the ester carboxyl-reduced, and the product partially hydrolyzed. The resulting, partially O-methylated oligosaccharides were reduced and ethylated, and the mixture of products was fractionated by l.c. The l.c. fractions containing per-O-alkylated oligosaccharide-alditols were analyzed by e.i.-m.s. Pure per-O-alkylated oligosaccharide-alditols were also analyzed by ¹H-n.m.r. spectroscopy. The products obtained by base-catalyzed degradation and subsequent ethylation of the per-O-methylated polysaccharide were fractionated by l.c. The main product isolated was analyzed by e.i.-m.s., c.i.-m.s., and ¹H-n.m.r. spectroscopy. The results of these studies, in conjunction with results of analytical methods commonly used in the elucidation of polysaccharide structures, unambiguously characterized the primary glycosyl structure of the polysaccharide. Base-labile substituents, previously reported to be present in the polysaccharide, were not studied. Structure 1 revises, and complements, previously reported structures.