Biological processes in organised media

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.     

Uphill transport induced by counterflow

1. In a membrane transport system containing a mobile carrier with affinities for two substrates a concentration gradient with respect to one of the substrates under certain conditions is able to induce an "uphill" transport (against the concentration gradient) of the other. 2. In a kinetic treatment quantitative conditions for such a "flow-induced uphill transport" and some of its characteristics are derived. 3. Experimentally the uphill transport of labelled glucose induced by a concentration gradient for mannose or unlabelled glucose is demonstrated in the human red cell. 4. It is shown that the flow-induced uphill transport is a feature characteristic for mobile carrier systems only and is not to be expected in systems in which the substrate is bound to a fixed membrane component ("adsorption membrane"), although such a system may yield identical transport kinetics. Also with respect to Ussing's flux ratio the two systems are different, the adsorption membrane meeting Ussing's criterion, the carrier membrane not. 5. It is concluded that the transport system in the human red cells must contain a mobile carrier, identical for glucose and mannose.     

Amino acid transport systems in animal cells: Interrelations and energization

After summarizing the discrimination of the several transport systems of neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends to run forward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali-ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate-aspartate shuttle. After depletion of the energy reseves in the Ehrilich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restoration by pyruvate but in poor correlation with the restoration of alkali-ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly senstitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali-ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na⁺-independent system, as well as to one entering by the principal Na⁺-dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali-ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali-ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.     

Asymmetry and free energy transduction in biology

This paper is an extension of our earlier theoretical studies on the relationship between kinetic asymmetry and free-energy transductions in biological systems induced by external fluctuations. In the first part of the paper, the asymmetry conditions necessary for external-noise-induced free-energy transductions to occur are derived for a special cyclic, four-state model in which only one reaction step is perturbed by the fluctuations. The results can be used to explain the earlier findings that asymmetry in rate constants was not required in the uphill transport of ligands induced by externally fluctuating the ligand concentrations. In the second part of the paper, the coupling between two enzyme systems through direct enzyme-enzyme inter-actions is studied. The existence of kinetic asymmetry in both the driving and the driven enzyme systems is found necessary for coupling and free-energy transductions to occur.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes

An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non-activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 microM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red-Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl-Sepharose and further chromatographic separation by red-Sepharose and FPLC using Mono-Q and phenyl-Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.     

Serum-induced net K+ influx performed by the diuretic-sensitive transport system in quiescent NIH 3T3 mouse fibroblasts

In serum deprived NIH 3T3 mouse cells the diuretic-sensitive transport system performs K+ self-exchange. The addition of serum which stimulates cell proliferation induces a net influx of K+, carried out by the diuretic-sensitive transport system. Thus, serum growth factors appear to induce a change in the mechanism of action of the diuretic-sensitive transporter from K+ self-exchange to an uphill transport pumping K+ into the cell. I propose here that this uphill uptake of K+ contributes to the increase of intracellular K+ content, found in the early G1 phase of the cell cycle.     

A new electrophoretic-autoradiographic method for the visual detection of phosphotransferases

In this paper we present the details of a slab acrylamide, lanthanum precipitation, autoradiographic technique and show it to be useful for the visualization of at least four different enzymes. We believe that with appropriate separation conditions and reaction mixtures this technique could be extended to a larger number of enzymes, theoretically all those whose isotopically labeled product could be specifically precipitated within the matrix of a polyacrylamide gel. It will be interesting to use this technique with other gel buffer systems, particularly those with a lower pH that have recently been reported (21). In addition, it might be useful to combine it with isoelectric focusing in slab gels (10). The technique would appear to be particularly useful for phosphotransferases and, to date, it has been applied to thymidine kinase and adenosine kinase with encouraging results. Work with other enzymes, and adapting the technique to starch gels, is also in progress.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

tight coupling of monosaccharide transport and metabolism inRhodotorula gracilis

A variety of agents have been applied to separate the uphill, metabolically linked transport of monosaccharides from facilitated diffusion inRhodotorula gracilis. The substances used were iodoacetamide, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, sodium azide, sodium fluoride and heavy-metal cations, including Cd²⁺, Hg²⁺, Cu²⁺, UO²⁺ ₂, La³⁺ and Th⁴⁺. No agent examined led to such separation. There was always either a complete inhibition of both cell metabolism (or uncoupling of oxidative phosphorylation) and the transport mechanism or the transport of sugars uphill persisted (when the metabolism was only partly inhibited). It is concluded that the monosaccharide transporting system inRhodotorula gracilis is tightly coupled to cell metabolism and cannot operate without sufficient energy supply even in cases when no uphill movement is involved.     

乳酸脱氢酶与酯酶同工酶同板染色法

介绍一种在同一块凝胶板上染乳酸脱氢酶(LDH)与酯酶(EST)的染色方法. 该同板染色法利用两种同工酶显色反应互不干扰和颜色不同的特点, 先染LDH, 后染EST, 可以在同一块胶板上得到两种同工酶清晰的酶带, 每一种酶的酶带与单板染色的酶带完全一样. 这种染色法, 能节省同工酶分析所需的试剂、时间和经费, 也便于样品的鉴定与比较, 是一种经济有效的方法. 此方法, 同样适用于苹果酸脱氢酶(MDH)与酯酶等同工酶的同板染色.     

Nature and properties of hexitol transport systems in Escherichia coli.

In Escherichia coli K-12 the naturally occurring hexitols D-mannitol, D-glucitol, and galactitol are taken up and phosphorylated via three distinct transport systems by a mechanism called either group translocation or vectorial phosphorylation. For every system, a membrane-bound enzyme II-complex of the phosphoenolpyruvate-dependent phosphotransferase system has been found, each requiring phosphoenolpyruvate, enzyme I, and HPr or alternatively P-HPr as the phosphate donor. Cells with a constitutive synthesis of all hexitol transport systems but with low P-HPr levels have very low transport and phosphorylating activities in vivo, although 40 to 90% of the enzyme II-complex activities are detected in cell extracts of such mutants. No indications for additional hexitol transport systems, especially for systems able to transport and accumulate free hexitols as in Klebsiella aerogenes, have been found. Substrate Km, and Vmax of the three transport systems for several hexitols and hexitol analogues have been determined by growth rates, transport activities, and in vitro phosphorylating activities. Each system was found to take up several hexitols, but only one hexitol serves as the inducer. This inducer invariably is the substrate with the highest affinity. Since bacterial transport systems, as a general rule, seem to have a relatively broad substrate specificity, in contrast to a more restricted inducer specificity, we propose to name the system inducible by D-mannitol and coded by the gene mtlA the D-mannitol transport system, the system inducible by D-glucitol and coded by gutA the D-glucitol transport system, and the system inducible by galactitol and coded by gatA the galactitol transport system.     

Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.

Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find alpha-AIB to be a poor competitor of alanine and phenylalanine uptake.     

Counter-transport mediated by the lactose permease of Escherichia coli.

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of preloaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H+ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide m chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP+). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP+ had no inhibitory effect on the overshoot at pH 6 and below.     

Theoretical analysis of the significance of whether or not enzymes or transport systems in structured media follow Michaelis-Menten kinetics

Pure Michaelis-Menten enzymes have been studied (i.e., enzymes with a hyperbolic (S, V) behavior in a well-stirred solution). When such enzymes are associated with a structure in vitro, even in the simplest conceivable form (immobilization in a homogeneous gel), they can produce enzymic or transport reactions with many different kinetics (Michaelis-Menten, sigmoidal, dual-phasic, etc.). Therefore, when structured enzyme or transport processes in vivo have sigmoidal kinetics, it is not proof that the corresponding proteins are allosteric. In same manner, when the apparent kinetics are dual-phasic, it is not proof that two enzyme, or transport systems, coexist.     

Protein turnover in the cytoplasmic transport system within an insect ovary--a clue to the mechanism of microtubule-associated transport

The movement of radioactively labelled polypeptides into the microtubule-associated transport channels in the ovaries of a hemipteran insect has been analysed using SDS-polyacrylamide slab gel electrophoresis and fluorography. The patterns of label suggest that the microtubules which pack the transport channels form a relatively static cytoskeleton while other components move independently from them along the channels. As well as illustrating the functional organisation of microtubule-associated transport in this system our studies of labelled proteins have also provided clues as to the mechanism of transport itself.     

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).     

ATP synthesis driven by proton transport in F1F0-ATP synthase

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.     

Elucidation of an a and L system for amino acid transport in the human lymphoblast using a membrane filtration technique

Summary Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid. Using this procedure we have characterized two transport systems for the neutral amino acids; an A and an L system, which are similar but not identical to the A and L systems characterized in rodent cell lines. These A and L systems have characteristically lower Km's and Vm's for alanine and phenylalanine, when compared to rodent cell lines. In addition, we find α-AIB to be a poor competitor of alanine and phenylalanine uptake. This work was supported by Grant No. CA18644, awarded by the National Cancer Institute, Department of Health, Education and Welfare, and from a grant from the National Science Foundation under Grant No. PCM 76-24328.