Conformational stabilization of enzymes in covalent catalysis.

The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pH-induced structural transitions to inert forms that occur in the free enzyme species and other forms not covalently substituted.     

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

Quantitative determination of amino acid racemization in heat-alkali-treated melanotropins: Implications for peptide hormone structure-function studies

The treatment of frog skins (in vitro) and frogs (in vivo) with melanotropins that have been heated briefly in aqueous alkali resulted in prolonged skin darkening. It has been postulated that this increase in melanotropic activity is related to the partial racemization of amino acid residues of the melanotropins. Quantitative determination of the extent of racemization of eight amino acids (Val, Pro, Met, Phe, Glu, Asp, Nle, Ser) present in α-melanotropin (α-MSH), [4-norleucine]-α-MSH, β_porcine-melanotropin (β_p-MSH), and [7-norleucine]-β_p-MSH after brief heat-alkali treatment, was accomplished using a high-resolution gas chromatographic technique. Phenylalanine-7 in α-MSH and [4-norleucine]-α-MSH and phenylalanine-10 in β_p-MSH and [7-norleucine]-β_p-MSH were found to be partially racemized to a greater extent than expected. Other amino acid residues were also racemized to unexpected degrees. The subsequent synthesis of an α-MSH analog containing d-phenylalanine-7, [4-norleucine, 7-d-phenylalanine]-α-MSH, resulted in a highly potent melanotropin with ultralong biological activity, as determined by frog skin bioassay, stimulation of mouse melanoma cell tyrosinase activity, and activation of mouse melanoma adenylate cyclase.     

Synthesis and quantitation of glucitollysine, a glycosylated amino acid elevated in proteins from diabetics

A conceptually simple method of vertical gel electrophoresis is presented. It involves the use of pliable plastic envelopes to house individual gels and their constituent buffer reservoirs and electrical systems completely submersed in coolant separate from other gels. In conjunction with a common cooling tank this envelope technique provides unusual versatility for running a variety of different type gels simultaneously.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

The analysis of nanogram levels of free amino acids by reverse-phase high-pressure liquid chromatography.

A simple method and apparatus are described for the efficient recovery of proteins from sodium dodecyl sulfate-polyacrylamide gel systems after electrophoretic resolution. This procedure provides for high yields of proteins which are free of sodium dodecyl sulfate and in certain cases, exhibit significant levels of biological activity.     

Synthesis and first in vitro cytotoxicity studies of bis(2-chloroethyl) amino group containing polymers. Pharmacologically active polymers: 22

Monomers containing the cytotoxic bis(2-chloroethyl)amino group (nor-nitrogen mustard), linked in deactivated form via urethane and O-acylated hydroxamic acid bonds to polymerizable methacrylic acid derivatives, have been prepared. Besides the homopolymerization, these monomers were copolymerized with hydrophilic comonomers to obtain water-soluble polymers. The linkages used are expected to undergo intracellular enzymatic or hydrolytic cleavage, releasing the cytotoxic bis(2-chloroethyl)amino moiety from the polymeric carrier. Preliminary in vitro cytotoxicity data for the polymers against three rat and mouse experimental tumour lines (Walker 256 carcinosarcoma, L1210 murine leukaemia and ADJ/PC6A plasma cell tumour) are reported. An approximately 10² fold difference in cytotoxic potency was found, depending on the type of the cleavable spacer group.     

Degradation of polysaccharides containing uronic acid residues

A method for the selective degradation of polysaccharides containing uronic acid residues is described. It involves methylation of hydroxyl and carboxyl groups, base-catalysed elimination, and mild hydrolysis with acid. The degraded product is etherified with trideuteriomethyl or ethyl groups and hydrolysed, and the resulting mixture of etherified sugars is analysed, as the alditol acetates, by g.l.c.—m.s. Comparison of this analysis with the methylation analysis of the original polysaccharide gives information on the nature of the sugar residues on either side of the uronic acid residue.     

Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein.

When ¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of ¹²⁵I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound ¹²⁵I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of ¹²⁵I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

Acid phosphatases of rabbit spermatozoa: II. Partial purification and biochemical characterization of the multiple forms of rabbit spermatozoan acid phosphatase

Five acid phosphatases, S4, S3, S2, Szn and S1 (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) of ejaculated rabbit spermatozoa were either partially purified by DEAE-Sephadex column chromatography or prepared by specific extraction methods.The pH optimum of S4 was 6.0–6.5 in acetate buffer and 7.0 in Tris-HCl buffer; the pH optima of S3, S2, Szn, and S1 were 4.5, 5.5., 6.0 and 5.2, respectively, in acetate buffer. The apparent molecular weights of S3, S, Szn and S1, determined by disc gel electrophoresis, were 123 000, 86 000, 64 000 and 45 000–49 000, respectively. Incubation with neuraminidase did not alter the electrophoretic mobilities of any of the enzymes.Ten natural phosphoric esters were tested as substrates. S4 preferentially hydrolyzed ATP, ADP, PP_i and 3′-AMP. S3 hydrolyzed only β-glycerophosphate and glucose 6-phosphate to a significant extent. S2 hydrolyzed β-glycerophosphate, glucose 1-phosphate, the phosphoproteins, casein and phosvitin. S1 hydrolyzed ADP and β-glycerophosphate most readily. Szn may be an ATPase since it exhibits very high Zn²⁺-stimulated against ATP.These characteristics combined with the effects of NaF, ZnCl₂, l-(+)-tartaric acid, and formaldehyde on the activity of each partially purified enzyme with α-naphthyl phosphate as substrate indicate that these phosphatases are structurally and functionally different.     

Tissue difference in gamma-glutamyl transpeptidase attributed to sialic acid content.

Gamma-glutamyl transpeptidase (GGT) was extracted from the microsomal fraction of various bovine tissues and partially purified. Purified enzymes demonstrated different mobilities toward the anode in polyacrylamide gel electrophoresis in 0.5% Emulphogene BC720, pH 7.5. The ciliary-body GGT migrated fastest, while the brain enzyme was electrophoresed most slowly. The apparent Km values (Km′) of GGT for L-gamma-glutamyl-p-nitroanilide were 1.4–2.0 mM when assayed with glycylglycine as the gamma-glutamyl acceptor. After neuraminidase treatment, electrophoretic mobility was decreased considerably for all enzyme preparations, compatibly with the removal of negatively charged sialic-acid residues. The Km′ values of the enzyme were not affected by the hydrolytic treatment. Electrophoresis of digested enzymes showed essentially identical mobilities. From these results we conclude that tissue differences in GGT are attributable to the varying extent of sialylation of enzyme.     

Mechanism and stereospecificity of alpha-hydroxylation of lignoceric acid in rat brain.

Cerebronic acid (2-hydroxytetracosanoic acid) is the major fatty acid component of cerebrosides and sulfatides in mammalian brain. Our previous communication demonstrated the synthesis of cerebronic acid from lignoceric acid (tetracosanoic acid) by a rat brain preparation in the presence of molecular oxygen and a reduced pyridine nucleotide (Hoshi, M., and Kishimoto, Y. (1973) J. Biol. Chem., 248, 4123–4130). The present'studies on the conversion of (RS)-[2-³H]-, (RS)-[3-³H]-, (R)-[2-³H]-, and (S)-[2-³H]lignoceric acids to cerebronic acid by rat brain preparations establish that the pro-R hydrogen at the α-carbon of lignoceric acid is replaced by a hydroxyl group with overall retention of configuration.     

The hemoglobins of phoronopsis viridis, of the primitive invertebrate phylum phoronida: characterization and subunit structure.

The primitive invertebrate, Phoronopsis viridis, of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH₂-terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.     

Affinity binding of the cartilage proteoglycan protein-keratan sulfate core to immobilized hyaluronic acid.

The protein-keratan sulfate core of bovine nasal cartilage proteoglycan was purified by affinity chromatography on a column of immobilized hyaluronic acid. The hyaluronic acid was immobilized by reaction with a hydrazido-alkyl derivative of Sepharose in the presence of borohydride. Proteoglycan was digested with chondroitinase ABC and the entire mixture was passed over a column of the Sepharose-hyaluronic acid maintained at 4°C. After the digested chondroitin sulfate chains were washed from the column, the bound protein-keratan sulfate core was eluted with 4m guanidinium chloride. The protein-keratan sulfate core interacts with the affinity matrix through its hyaluronic acid binding site as shown by the inhibition of binding by free hyaluronic acid and hyaluronic acid decasaccharide.     

The role of the superoxide anion in the xanthine oxidase-induced autoxidation of linoleic acid.

A fluorescent analogue, palmitoyl-?CoA was shown to have a fluorescence lifetime (19.5 nsec.), polarization and absorption and emission characteristics useful for studying interactions with enzymes and with model membranes. The fluorescence lifetime was found to be wavelength dependent. The analogue was a better inhibitor (50% inhibition at ～ 0.2 μM) than palmitoyl-CoA (50% inhibition at 0.5 μM) when bound to mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxido reductase E.C.l.l.137). The fluorescence depolarization when bound to this enzyme was less than that observed for binding to bovine serum albumin suggesting some mobility of the chromophore while bound. The changes in polarization upon titration with phosphatidylcholine (egg) vesicles were consistent with a partition of palmitoyl-(1,N⁶etheno)CoA between vesicles and malate dehydrogenase. Such partition may have physiological consequences.     

The separation and amino acid analysis of collagen crosslinks on an extended basic ion-exchange column

The major reducible crosslinks found in collagen were separated and analyzed on an extended basic amino acid analyzer column. Reaction with ninhydrin allows the direct analysis of collagen crosslinks, including hydroxyaldol-histidine, a naturally occurring, nonreducible crosslink. In addition to known crosslinks, direct amino acid analysis of tissue hydrolysates reveals the presence of an unknown, ninhydrin-reactive component, in both NaB³H₄-reduced and unreduced collagenous tissues. Initial fractionation of hydrolysates on a Bio-Gel P-2 gel filtration column provides partial separaton of amino acids and crosslinks and enables more direct analysis of the crosslinks present in the samples, as well as detecting potential new crosslinks. The results also show that, prior to NaB³H₄ reduction, substantial amounts of known crosslinks are normally present in bovine skin and bone.