alpha-Galactosidase (melibiase) from Saccharomyces carlsbergensis: structrual and kinetic properties.

This report describes structural and kinetic properties of the purified α-galactosidase from Saccharomyces carlsbergensis. This galactosidase has many similar properties to other exocellular enzymes in yeast which have been reported. Its molecular weight of 300,000 is comparable; it has similar carbohydrate content (57%) and amino acid and carbohydrate composition. That is, 35% of its amino acid residues can be accounted for by threonine, serine, and aspartic acid. Its carbohydrate composition is primarily mannose (90–95%) with approximately 7% glucose and 1% glucosamine. The enzyme is very stable to both acidic and alkaline conditions as well as to heating to 50 °C. α-Galactosidase remains active after incubation with as much as 1% sodium dodecyl sulfate at 30 °C. However, the enzyme is denatured with urea and guanidine hydrochloride. The loss of activity is proportional to the urea concentration, the nondenatured enzyme being responsible for the remaining activity. Inactivation by urea is partially reversible. With urea or 60 °C heat denaturation, the enzyme dissociates into two types of subunits as revealed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Thus, α-galactosidase is the first external enzyme from yeast in which an oligomeric structure is reported. The enzyme catalyzes the hydrolysis of p-nitrophenyl-α-d-galactoside, melibiose, and raffinose with similar pH optima and V_max. However, the affinity is 20-fold lower for raffinose than for the other two substrates. Sugars having the same configuration in carbons 2, 3, and 4 as galactose competitively inhibit the enzyme.     

Vasoactive intestinal peptide (VIP) from chicken: Synthesis and properties of the C-terminal hendecapeptide

The hendecapeptide, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Thr-NH₂, corresponding to sequence 18–28 of chicken vasoactive intestinal peptide (VIP), was synthesized stepwise, starting with the C-terminal residue. The in situ technique was applied; o-nitrophenyl esters and p-nitrophenyl esters were used for acylation. The product was compared with, and found indistinguishable from, the C-terminal cyanogen bromide fragment of natural chicken-VIP. Some pharmacological properties of the hendecapeptide were also determined. In two separate experiments, the chain of the hendecapeptide was further lengthened to encompass residues 14–28 of chicken-VIP but with leucine and norleucine in place of methionine in position 17. The two pentadecapeptides showed biological activities comparable to those of the C-terminal pentadecapeptide fragment of porcine VIP or its 17-norleucine analog.     

Human liver cytosolic malate dehydrogenase: Purification,kinetic properties,and role in ethanol metabolism

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 ± 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (μm) of the kinetic parameters were obtained at pH 7.4 and 37 °C: K_a, 17; K_ia, 3.6; K_b, 51; K_ib, 68; K_p, 770; K_ip, 10,700; K_q, 42; K_iq, 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD⁺, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 μmol/min/g wet wt of liver for oxalacetate reduction and 11.2 μmol/min/g liver for malate oxidation at pH 7.4 and 37 °C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ redox state during ethanol metabolism in human liver.     

Cytoskeletal and contractile structures in bovine lens cell differentiation

Bovine lenses from animals of different ages were separated into two epithelial sections, a cortical region and the lens nucleus. Both the 10000 g supernatant fraction and pellet of these sections were analysed by electrophoresis in SDS-containing polyacrylamide gels. When comparing total protein patterns of the cytoskeletal preparations from the different parts of lenses of different ages a decrease in the amount of vimentin, the protein subunit of lens intermediate-sized filaments (IF), was observed upon lens cell differentiation and aging. Amounts of monomeric (G) and filamentous (F) actin in the different stages of lens cell differentiation were quantitated using the DNase I inhibition technique. A significant increase in the relative amount of F-actin was observed upon fibre cell formation. A slight, but significant increase in the total amount of actin relative to the total amount of cellular protein was observed when passing from the central part of the lens epithelium to the epithelial cells in the elongation zone. In the fibre cells the amount of total actin decreased from cortex to nucleus. A possible function of microfilament-assembly in the process of lens cell differentiation is suggested.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

Isolation and properties of an aminopeptidase from Bacillus licheniformis

An extracellular aminopeptidase, purified 465-fold from culture filtrates of Bacillus licheniformis, was found to be a metalloenzyme consisting of a single peptide chain. Sedimentation equilibrium yielded a molecular weight of 43,270 and two polyacrylamide electrophoretic procedures gave values of 37,500 and 36,000, respectively. The activity of the enzyme was inhibited severely by 1,10-phenanthroline and to a lesser extent by EDTA, cyanide, and fluoride. The addition of Co²⁺ ions greatly stimulated enzymatic activity, but analysis of the purified enzyme revealed the presence of zinc, not cobalt, in stoichiometric quantities. Moreover, the ratio of zinc to protein was found to increase during fractionation, reaching a final value corresponding to 1 g-atom/mol. The aminopeptidase possessed characteristics of a euglobulin, sparingly soluble in water and dilute buffer solutions, but soluble in buffers containing higher concentrations of salts. Both activity and pH optimum were substantially influenced by ionic strength; as the latter was increased over the range from 0.01 to 0.1, activity increased and the pH optimum was shifted to more acidic values. Enzymatic activity was affected by the identity of the buffer, being markedly greater in Tris-HCl than in sodium barbital and strongly inhibited by phosphate. The Bacillus aminopeptidase hydrolyzed substrates with unsubstituted amino groups of the l configuration, including dipeptides, aminoacylnaphthylamides, and amino acid amides.     

S-adenosylhomocysteine hydrolase from hamster liver: Purification and kinetic properties

3-Deazaadenosine is both an inhibitor of and a substrate for S-adenosylhomocysteine hydrolase. Its administration to rats results in the accumulation of both S-adenosylhomocysteine and 3-deazaadenosylhomocysteine in the liver and other tissues. In hamsters, however, the administration of 3-deazaadenosine results only in the accumulation of 3-deazaadenosylhomocysteine (P. K. Chiang and G. L. Cantoni (1979) Biochem. Pharmacol. 28, 1897). In order to investigate the possible reasons for this difference, S-adenosylhomocysteine hydrolase from hamster liver has been purified to homogeneity and some of its kinetic and physical parameters have been determined. The molecular weight of the native enzyme is 200,000 with a subunit molecular weight of 48,000. The K_m's for adenosine and 3-deazaadenosine are about 1.0 μm, and the V_max's are also similar. The K_m for S-adenosylhomocysteine is 1.0 μm, or more than 10 times smaller than the K_m of the rat liver enzyme. This difference in K_m value may explain the differences in the response of rat and hamster liver to the administration of 3-deazaadenosine. S-Adenosylhomocysteine hydrolase from hamster liver exhibits an interesting kinetic property in that its activity can be affected bimodally by either adenosine or adenosine Anal.ogs. At very low concentrations of these analogs, the activity of S-adenosylhomocysteine hydrolase can be stimulated by 10–30%, and at higher concentrations these same analogs become competitive inhibitors.     

Labeling of bovine corpus luteal plasma membrane human chorionic gonadotropin or luteinizing hormone (hCG/LH) receptor and its purification and properties.

A method has been developed for labeling receptors for human chorionic gonadotropin or luteinizing hormone ($\text{hCG}\text{LH}$) present on bovine corpus luteal plasma membranes. It consists of four steps: (a) protection of the receptor by treating the plasma membranes with hCG; (b) iodination of the membranes with KI using glucose, glucose oxidase, and lactoperoxidase; (c) unmasking the receptor with either 2 m NaCl, 1 m guanidine hydrochloride, or rabbit anti-hCG; and (d) reiodination of the membranes using Na¹³¹I. After solubilization by successive treatments with Sepharose-concanavalin A and Sepharose-hCG and finally by preparative disc electrophoresis, the resulting purified receptor after electrophoresis in polyacrylamide gel showed a single radioactive band containing receptor activity. This highly purified receptor is fairly stable and retains its hormonal specificity, binding affinity, and pH optimum. It was observed that the receptor alone or as a complex with the hormone tends to aggregate. The receptorhormone complex does not dissociate during polyacrylamide-gel electrophoresis.     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Synthesis and properties of the cyclopentapeptide desthiomalformin

Desthiomalformin, cyclo-d-alanyl-d-alanyl-l-valyl-d-leucyl-l-isoleucyl, was synthesized by conventional methods. Cyclization of the open chain intermediate through the azide produced the cyclopentapeptide in short reaction time and in high yield and was not accompanied by cyclodimerization. The extreme readiness of the pentapeptide azide to form a cyclic structure can be attributed to the presence of alternating d and l residues in the sequence, a feature that should result in reverse turns stabilized by intramolecular hydrogen bonds. Thus the open chain intermediate could have a preferred cyclic conformation.Desthiomalformin has high thermal stability; it can be sublimed in vacuo without decomposition. It lacks, however, the biological activity of its parent compound, malformin.     

Methylated cycloamyloses (cyclodextrins) and their inclusion properties

2,3,6-Tri-O-methyl and 2,6-di-O-methyl derivatives of cyclohexa- and cyclo-hepta-amylose (2,3,6-tri-O-methyl- and 2,6-di-O-methyl-α- and -β-cyclodextrin) have been prepared and shown to be versatile complexing agents. Their complexes in aqueous solution are usually more stable than the corresponding complexes of un-substituted cycloamyloses. The methylated cycloamyloses also form crystalline complexes, the stability of which depends on the size and shape of the guest molecule. The decomposition temperature of the crystalline complexes with homologous n-alkanes, which is relatively high increases with increasing chain-length of the hydro-carbon. The shift of the i.r. carbonyl band of oleic acid in its solid complex with methylated α-cyclodextrin probably reflects inclusion of the fatty acid in the mono-meric form. The methylated cycloamyloses, when used as the stationary phases for g.l.c. or dissolved in a conventional stationary phase, affect the retention times of organic compounds in a manner which suggests that inclusion phenomena are operative.     

Synthesis and characterization of tertiary phosphine Pd(0) complexes

The synthesis of PdL_n complexes (L = tertiary phosphine; n = 2,3,4) is reported. The coordination number results to be a function of the steric hindrance of the phosphine. Two-coordinate, 14-electron complexes, have been isolated with bulky phosphines (PPrⁱ₃, P (cyclohexyl)₃, PBu^t₂Ph). The behaviour in solution of the PdL_n complexes has been studied by ¹³C NMR spectroscopy.     

Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP)

Dispersed cells of the bovine adrenal medulla express immunoreactive vasoactive intestinal polypeptide (VIP) after 24 hours in culture, although VIP could not be detected in extracts of bovine adrenal medulla or cortex. Immunoreactive VIP eluted from a reversed-phrase chromatography column with the same retention time as authentic porcine VIP_1–28. VIP in chromaffin cells in culture appears to be contained in a secretory granule pool, since it, like methionine-enkephalin (met-enk) was released into the medium after exposure of cells to nicotine, carbachol, veratridine and elevated potassium in a dose-dependent manner. Doseresponse curves for VIP and enkephalin release by the above secretagogues were similar but not identical. Enkephalins and VIP may either be contained in separate subpopulations of chromaffin cells or co-stored in the same cells.     

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.     

Purification and properties of NADP+:Isocitrate dehydrogenase from lactating bovine mammary gland

NADP⁺:isocitrate dehydrogenase has been purified to homogeneity from lactating bovine mammary gland. Purification was achieved through the use of affinity and DEAE-cellulose chromatography. The isolated enzyme gives one band when stained for protein or enzyme activity on discontinuous alkaline gel electrophoresis. The enzyme has a molecular weight of 55,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and a Stokes radius of 4.1 nm as measured by gel chromatography. The enzyme will not use NAD⁺ in place of NADP⁺ and has an absolute requirement for divalent cations. The apparent K_m values for dl-isocitrate, Mn²⁺, and NADP⁺ were found to be 8, 6, and 11 μm, respectively. The Mn²⁺-d_s-isocitrate complex is the most likely substrate for the mammary enzyme with a K_m of 3 μm. The properties of mammary NADP⁺:isocitrate dehydrogenase are compared with those of the homologous enzymes from pig heart and bovine liver, and its characteristics are discussed with respect to the function of the enzyme in lactating mammary gland.     

Chitin and chitinase: A kinetic model

A model is described for the action of insect molting chitinase on chitin microfibrils in cuticle. The model reconciles the disparate structures proposed for chitin in the literature. It also accounts for the kinetic characteristics of molting fluid chitinase insofar as known from in vitro studies, viz. positive co-operativity of possibly three catalytic sites, complexity, and processivity. These have hitherto been difficult to account for in vivo, given the arrangement of chitin in anhydrous microfibrils in arthropod cuticle.     

Dissociation and oxygen equilibrium properties of earthworm (Pheretima hilgendorfi) hemoglobin

The dissociation and oxygen equilibrium properties of the purified hemoglobin and whole blood obtained from the earthworm Pheretima hilgendorfi were compared. Above pH 8.0, P1/2's were higher in the purified hemoglobin than in whole blood, while below pH 8.0, nearly identical P1/2's were observed in both materials and P1/2 was at a maximum at pH 6.5. The values of n1/2 were higher in whole blood than in the purified hemoglobin at alkaline pH. The maximum values of n1/2 were observed around pH 8.1 in the purified hemoglobin and pH 8.7 in whole blood, and the values were 5.3 and 9.5, respectively. In the purified hemoglobin, a small amount of dissociation component was already observed at pH 8.0, while in whole blood, no dissociation occurred up to pH 9.1. Dialysis of whole blood or addition of 10 mM EDTA to whole blood at alkaline pH induced the loss of the enhanced cooperativity, the increased oxygen affinity and the high stability of the 60 S whole molecule. These results strongly suggest that divalent cations are participating in the functional and dissociation properties of the whole blood of this species.