Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity.

Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to approximately 6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal approximately 50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH2-terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s-1 and 35 s-1, respectively, for kcat at saturating microtubules).     

Characterization of a microtubule-stimulated adenosinetriphosphatase activity associated with microtubule gelation-contraction

A microtubule-stimulated ATPase is associated with particles that are responsible for microtubule gelation-contraction in vitro. These particles have been proposed to be slow axonal transport, component a, particulates (SCAPs) [Weisenberg, R. C., Flynn, J. J., Gao, B., Awodi, S., Skee, F., Goodman, S., & Riederer, B. (1987) Science (Washington, D.C.) 238, 1119-1122]. The SCAP ATPase activity is stimulated approximately twofold by microtubules. The microtubule-stimulated ATPase activity correlates with the occurrence of microtubule gelation-contraction. Both microtubule-stimulated ATPase activity and microtubule gelation-contraction are inhibited by millimolar calcium, 0.3 M KCl plus 2 mM ethylenediaminetetraacetic acid (EDTA), 5 microM vanadate, and millimolar N-ethylmaleimide (NEM). Neither the ATPase activity nor microtubule gelation-contraction is affected by high magnesium concentrations (up to 8 mM) or by the anti-ATPase drugs ouabain, oligomycin, sodium azide, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Magnesium is required for both ATPase activity and microtubule gelation-contraction. Microtubule-stimulated hydrolysis of GTP, CTP, ITP, and UTP is less than 50% of ATP hydrolysis, and microtubule gelation-contraction is reduced in these nucleotides. On the basis of these results we propose that the microtubule-stimulated ATPase activity associated with SCAPs is a previously undescribed enzyme that is responsible for microtubule gelation-contraction in vitro and that is the likely motor for component a of slow axonal transport.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.     

A functional link between the dark Mg-ATPase activity and the light-induced enzymatic cascade in rod outer segments

The characterization of a light-induced scattering change in suspensions of rod fragments, which requires previous swelling of the disks by the dark Mg-ATPase described by Uhl et al. [FEBS Lett. 107, 317-322 (1979)] is reported here. Reconstitution experiments demonstrate that this signal is dependent on the presence of G-protein, GTP and cGMP phosphodiesterase. Fast reversal associated with regenerability requires in addition the presence of some protein(s) of the cytoplasm (probably the rhodopsin kinase) and ATP. The amount of excited rhodopsin which saturates the signal is the same as that which saturates the previously described 'dissociation signal' [Kühn et al. (1981) Proc. Natl Acad. Sci. USA 78, 6873-6877] associated with the formation of the phosphodiesterase activator G alpha GTP (alpha subunit of the G-protein with GTP bound). The kinetics of the signal is slightly slower than that of the dissociation signal and its amplitude is proportional to the extent of swelling of the disks. These results suggest that the interaction between G alpha GTP and the phosphodiesterase modifies some structural feature of the disks and provide evidence for the existence of a functional link between the dark Mg-ATPase and the light-induced enzymatic cascade.     

A two-step enzymatic glycosylation of polypeptides with complex N-glycans

A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.     

A kinetic study of the kinesin ATPase.

The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.     

Thick filaments of fish myosin and its actin-activated Mg-ATPase activity

1. Tilapia (Tilapia nilotica) myosin forms short, mini-filaments, and are easily disassembled upon addition of ATP showing no saturated activation in its actin-activated Mg-ATPase activity. 2. The presence of 5-10 mM MgCl2 allows tilapia myosin to form native thick-filaments and are resistant to ATP. 3. The rod portion of Tilapia myosin molecule is responsible for its characteristic filament forming ability. 4. The similar filament forming ability as Tilapia myosin was suggested for other fish myosins.     

Immobilized yeast cells with methanol oxidase activity: Preparation and enzymatic properties

Cells form the yeast Hansenula polymorpha (ATCC 26012) were successfully immobilized by entrapment in a polyacrylamide gel. The resulting gel showed high methanol oxidase activity especially after treatment with a detergent (CTAB). The enzymatic properties of the gel-entrapped cell were not very different from that of the soluble enzyme except that no inhibition was observed at high methanol concentration. In continuous reactors, the gel-entrapped cells showed a much higher stability than other enzyme preparations. The inactivation mechanism was investigated and proved to be the oxidation of essential SH group(s) of the methanol oxidase molecule by hydrogen peroxide. Treatment with β-mercaptoethanol prevented inactivation or regenerated activity.     

Evaluating kinetics of enzymatic saccharification of lignocellulose by fractal kinetic analysis

The effects of impacting factors, including cellulase loading, operation temperature, product glucose inhibition, and high solid pretreated biomass loading were examined systemically on the enzymatic saccharification of lignocellulose (dilute acid pretreated corn stover) in the presence and absence of tri-block copolymer L64 (also referred to polymeric nonionic surfactant). The complex kinetics of enzymatic saccharification of cellulose were subjected to fractal kinetic analysis based on a fractal kinetic model, which is described with fractal kinetic parameters of the rate constant and fractal exponent. The results indicate that glucose inhibition including high lignocellulose loading is indexed by decreasing rate constant while lignin inhibition and high operation temperature is indexed by increasing fractal exponent. The effect of a nonionic surfactant on the enzymatic saccharification of lignocellulose mainly contributed to the elimination of lignin inhibition by decreasing the corresponding fractal exponent. However, the effect of the nonionic surfactant on cellulase activity and stability was very limited.     

Studies on the transverse tubule membrane Mg-ATPase. Lectin-induced alterations of kinetic behavior

Transverse tubule (TT) membrane vesicles contain a very active Mg-ATPase (EC 3.6.1.3). Concanavalin A (ConA) and other lectins were found to activate the TT Mg-ATPase from chicken skeletal muscle up to 25-fold yielding specific activities greater than 800 mumol/h/mg. The sarcoplasmic reticulum Ca-ATPase and the sarcolemma Na,K-ATPase were unaffected by ConA. 125I-Labeled lectin binding to the TT membrane Mr 102,000 glycoprotein supports the contention that this protein is identical with or is intimately associated with the TT Mg-ATPase. The ATPase exhibited non-Michaelis-Menton kinetics with both apparent negative cooperativity (n = 0.723; S0.5, Mg-ATP = 14 microM) and substrate inhibition (Ki, Mg-ATP = 10.2 mM), both of which were eliminated in the presence of ConA. Under the same conditions, ConA also abolished the unusual temperature dependence and potent Triton X-100 inhibition. The similarities in ConA suppression of both Triton and substrate inhibition suggest that these ligands may be interacting through a non-catalytic site and that Triton is serving as a nucleotide-mimetic agent. The unique kinetic responses are consistent with a homotropic substrate modifier mechanism wherein the enzyme can be viewed as possessing a single catalytic and a single regulatory site on a single polypeptide chain. It is proposed that ConA interferes either with ligand interaction at a putative regulatory site or blocks communication between a regulatory site and the catalytic site. The possible nature of the regulatory site and its modulation by a ConA-like, endogenous, skeletal muscle lectin and their combined role in excitation-contraction coupling is discussed.     

Conformational and kinetic studies of synthetic polypeptides by NMR

The α-helix–coil transition of poly-L -leucine, poly-L -alanine and poly-L -methionine in chloroform–trifluoroacetic acid system was studied by nuclear magnetic resonance (NMR) and optical rotatory dispersion (ORD). The kinetics of the hydrogen–deuterium exchange in the peptide was also followed in these polymers by means of NMR. Two types of the NMR spectra and the hydrogen–deuterium exchange reaction were found, corresponding to the high and low molecular weight polypeptides. In high molecular weights, the NH and α-CH resonance lines gave single peaks and the hydrogen–deuterium exchange was expressed as a single first order reaction. In low molecular weights, the NH and α-CH lines were separated into two peaks, corresponding to helical and random-coiled states, respectively, and the exchange react ion was expressed as super-position of a very rapid exchange reaction in the random-coiled part and another slow exchange reaction of the first order in the helical part. These results suggest that the helix–coil interconversion of low molecular weight polypeptides has a longer relaxation time (? 4.5 × 10^?3 sec) than that of high molecular weight polypeptides.     

一株高产木聚糖酶真菌的筛选及酶学性质分析

从近百份土样中,首先筛选得到具有明显半纤维素水解活性透明圈的菌株564株,其中202株的木聚糖酶活力用96孔板筛选方法进行了验证。结果显示：基于96孔深孔板测定的酶活力与初筛测得的透明圈活力趋势一致。最终筛选得到一株高产木聚糖酶的菌株ECU2023,经核糖体rDNA内部转录间隔区的序列对比,鉴定为泡盛曲霉（Aspergillus awamori）。研究获得了其最佳培养条件：pH6．0,培养时间4d,C源为30～40g/L玉米芯粉。经过（NH4）2SO4沉淀和Superdex G-75凝胶过滤层析后,其中主要木聚糖酶成分的最适反应pH为6．0,最适反应温度为50℃。     

K, Mg-ATPase activity in guard cells of Commelina communis

Siliqua development was studied in the wild type line Landsberg erecta and the GA-sensitive mutant ga-1 of Arabidopsis thaliana (L.) Heynh. Reciprocal crosses between wild type and ga-l mutant, and self pollinations of either parent have shown that siliqua growth is determined by endogenous GAs originating from maternal tissues and embryo. The ga-1 mutant either self pollinated or cross-pollinated with wild type pollen showed reduced siliqua growth, which to a large extent could be overcome by exogenously applied GAs. The siliquae of the ga-1 mutant possessed very reduced ent -kaurene synthesizing capacity and no detectable endogenous GA-activity indicating an early block in the GA-biosynthetic pathway. Seed weight is not affected by GA-deficiency during development.     

Electrochemical assay of plasmin activity and its kinetic analysis

A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k_cat/K_m value was 0.063 μM⁻¹ s⁻¹. Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.     

亮白曲霉乳糖酶基因在毕赤酵母中的高效分泌表达及酶学性质研究

将克隆的亮白曲霉 (Aspergilluscandidus)乳糖酶基因lacb′插入到毕赤酵母 (Pichiapastoris)高效表达载体pPIC9中 ,与分泌信号肽序列α_因子融合 ,通过同源重组将lacb′整合到酵母染色体上。通过SDS_PAGE检测和表达产物的酶活性筛选 ,得到重组转化子 ,证明乳糖酶获得有效分泌和高效表达。表达的乳糖酶为糖蛋白 ,表观分子量为 130kD ,脱糖基后的蛋白分子量下降为 110kD。经过 5L小罐高密度发酵 ,重组酵母中酶蛋白表达量为 6mg mL发酵液 ,每毫升发酵液中乳糖酶的活力为 36 0 0U ,高于目前国内外报道的水平。进一步研究了表达产物的酶学性质 ,该酶最适pH为 5 . 2 ,最适反应温度 6 0℃ ,比活性为 70 6 . 5± 2 . 6U mg ,Km为 1 7mmol L ,Vmax为 3. 3μmol min。与米曲霉ATCC 2 0 4 2 3的乳糖酶相比 ,该乳糖酶具热稳定性强、比活性高、pH范围宽等特点。     

Conformation properties and enzymatic activity of pancreatic ribonuclease in soluble complexes with sodium dextran sulfate

Effect of complex formation with dextran sulfate (DS) (substitution degree 1.3, molecular mass 500 thousand) on RNAse enzymic activity. its spatial structure and conformation stability was studied. Hydrolytic activity of the enzyme in complex in inhibited already at small additions of DS, while the transferase one is changed only at a great excess of the polyelectrolyte. It has been shown by CD spectra that no notable conformation changes proceed in the enzyme during complex formation, although the enzyme turns destabilized to the denaturing effect of heat at the expense of strengthened interactions between DS and RNAse during its denaturation. Thus the inhibition of hydrolytic activity in the complex is primarily related to limitations for the formation of the enzyme-substrate complex on polyelectrolyte charged likely with the substrate, and not to the protein conformation changes.     

Structural and enzymatic properties of Ageritin,a novel metal-dependent ribotoxin-like protein with antitumor activity

Ageritin has been recently described as the first ribotoxin-like from Basidiomycota division (mushroom Agrocybe aegerita) with known antitumor activity (BBA 2017, 1861: 1113-1121). By investigating structural, catalytic and binding properties, we demonstrate that Ageritin is a unique ribotoxin-like protein. Indeed, typical of the ribotoxin family, it shows the specific ribonucleolytic activity against the ribosomal Sarcin-Ricin Loop in a rabbit reticulocytes assay. However, it displays several elements of novelty, as this activity is strongly metal-dependent and completely suppressed in the presence of EDTA, different from other representative members of the ribotoxin family. Consistently, we prove that Ageritin is able to bind magnesium ions with low micromolar affinity. We also show that Ageritin is significantly more stable than other ribotoxins in thermal and chemical denaturation experiments. These peculiar features make Ageritin the prototype of a new ribotoxin-like family present in basidiomycetes. Finally, given its high stability, this enzyme is a promising candidate as a new tool in immunoconjugates and nanoconstructs.