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The article presents information on several forthcoming research papers, which will be published in the coming issues of the European Journal of Biochemistry. Some of the topics of the papers are: lipolysis, biosynthesis, electron transfer, molecular weights, etc.

The article presents information on several forthcoming research papers, which will be published in the coming issues of the European Journal of Biochemistry. Some of the topics of the papers are: lipolysis, biosynthesis, electron transfer, molecular weights, etc.

A Rhesus monkey, serologically grouped as B, has been shown to excrete low-molecular-weight carbohydrate material in urine closely related to that found in human urine. Galactose feeding resulted in the excretion of a trisaccharide which was shown to be identical to the trisaccharide isolated from the urine of group B humans under the same conditions. Experiments in which [14C]galactose was administered both orally and via an intestinal vein demonstrated that the intestine is the site of biosynthesis. [ABSTRACT FROM AUTHOR]

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (08:K27-:H-) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 450% aqueous phenol at 65 °C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2-keto-3-deoxy-mannosulonic acid (0.8%). Polysaccharide II was obtained in 0.8–1% yield (based on dry bacteria). It contained 99.2% mannose. With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43 °C for polysaccharide I and 42 °C for polysaccharide II. Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas-liquid chromatography and mass spectrometry indicated that in both polysaccharides two-thirds of the mannose units were substituted at C-2 and one-third at C-3. These results were confirmed by periodate oxidation. From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry. It is concluded that the poIysaccharides I and II contain about 20 repeating units of α-mannosyl-1,2-α-mannosyl-1,2-α-mannose which are joined through &alph;-1,3 linkages. [ABSTRACT FROM AUTHOR]

The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA · RNA hybrid duplexes and DNA · DNA partial duplexes prepared by polymerization of fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme. [ABSTRACT FROM AUTHOR]

I. This paper describes some features of the protein synthesis in isolated rat liver mitochondria and reports on the stability of the product of mitochondrial protein synthesis in vitro. 2. Chase experiments performed after a 30-min pulse of [14C]leucine indicated that a large part of the product is degraded to an acid-soluble form (labile product) almost as rapidly as it is terminated and released from the mitoribosome. Only a small proportion of the completed product (between 20 and 40 %)is conversed in acid-insoluble form (stable product). 2. Both labile and stable products have an exceptional degree of hydrophobicity, shown by their marked resistance to acid and alkaline hydrolysis and by their solubility in organic solvents. 4. A 14C-labelled peptide with an apparent molecular weight of 12000-13000, as assessed by gel electrophoresis in the presence of dodecylsulfate, is released after heat treatment of mitoribosomes isolated from mitochondria pulsed with [14C]leucine. [ABSTRACT FROM AUTHOR]

Helix pomatia β-haemocyanin was studied in solution by small-angle X-ray scattering. The following molecular parameters were determined: molecular weight := 9.02 × 106, volume = 14000 nm3, radius of gyration = 18.4 nm, radius of the spherical subunits = 2.5 ± 0.2 nm. With these data, and with information of dissociation products described in a former paper, a model of the molecule was built whose theoretical scattering curve showed good agreement with the experimental one. The model consists of 160 spherical subunits of a radius of 2.5 nm; 12 rings each built up of 10 spheres form the outer wall of a hollow cylinder; 20 subunits are situated at the inner side of each end. [ABSTRACT FROM AUTHOR]

Various observations cited at the beginning of this paper and in the Discussion suggested that phalloidine, a poisonous constituent of the green mushroom Amanita phalloides, would not be toxic by itself, but would be rendered toxic previously in the liver. Using a tritiated toxic derivative of phalloidine ([³H]desmethylphalloin) we found that: (a) in rats, 60% of the administered substance are excreted unchanged in the urine within the first day. A second radioactive substance of low concentration, moving more slowly in paper chromatography turned out as a product of the self-degradation of [³desmethylphalloin; (b) respectively 98% and 99.9% of the radioactivity were extracted with methanol from homogenates of livers of rats or mice which had received [³H]desmethylphalloin 1 to 2 hours before. Aqueous homogenates of liver of poisoned rats also contained radioactivity firmly bound to a high molecular weight substance as shown by gel chromatography. The radioactive substance was totally removed from the carrier presumably ribosomal material, by adding methanol, and was identified as [³]desmethylphalloin; (e) [³H]desmethylphalloin was not metabolized on incubation with oxygen and rat liver ribosomes in a NADPH-regenerating system. A radioactive substance extracted from liver microsomes of a poisoned rat also proved to be original toxin. As a result of these experiments it can be concluded that [³H]desmethylphalloin and most probably also phalloidine are not metabolized in the livers of rats and mice. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]

A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described. Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature. Yeast phosphoglyceromutase contains four highly similar, probably identical subunits of molecular weight 28000, a conclusion based on the following observations. Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge. Cyanogen bromide cleavage and tryptic digestion of the enzyme yield the number of peptides expected for identical subunits from the amino acid composition analysis. The purified phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate and 3-phosphoglycerate. It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3-bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction [Sasaki, R. et al. (1971) Biochim. Biophys. Acta, 227, 584–594, 595–607]. Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

N-Acetylglycosamine kinase from the human gastric mucous membrane was purified about 170-fold. The purified enzyme is specific for N-acetylglucosamine with slight phosphorylation of N-acetylmannosamine. The aminosugar product of the enzymatic reaction was identified as N-acetylglucosamine 6-phosphate. The Km for N-acetylglucosamine is 0.11 mM and for ATP 3.03 mM. The purified enzyme is stable at +4 °C for three days, labile to dialysis and irreversibly inhibited by p-chloromercuribenzoate. The molecular weight of the enzyme was 77000. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from chicken gizzard muscle myosin. After S-carboxymethylation or performic acid oxidation, two light chain components (20 000-M[subr] and 17 000-M[subr] chains) were isolated by chromatography on a column of DEAE-cellulose in the presence of 4 M urea. Tryptic peptides of the S-carboxymethylated 20 000-M[subr] chain were isolated, and their sequences were determined. The alignment of these tryptic peptides in the chain was deduced from the amino acid compositions and from the partial sequences of peptic peptides of the oxidized protein. The established sequence consists of 171 amino acids and its calculated molecular weight is 19692. Comparing the sequence with those of L-2 chains from chicken and rabbit skeletal muscle myosins, 81 and 78 amino acid substitutions were recognized, respectively, including insertions and/or deletions. [ABSTRACT FROM AUTHOR]

Describes an application of two-dimensional, nuclear magnetic resonance techniques for the study of proteins. Amino acid residue; Molecular weight; Glycyl residue spin systems; Spin-spin coupling constants; Protein conformation.

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 °C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-I 5R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 °C it synthesizes a ‘cryptic’ protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30°C). The intracellular proteases from the wild type and from ts-15 grown at 30°C and 42°C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30°C was 64000–65000 in the absence of sodium dodecylsulfate and 31000–32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42°C showed the same molecular weight of 32000–34000 in the presence or in the absence of sodium dodecyisulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form. [ABSTRACT FROM AUTHOR]

In a recent paper in this journal it was shown that human ceruloplasmin contains a single continuous polypeptide chain composed of about 1050 amino acid residues. Together with the carbohydrate this accounts for the molecular weight of the native protein 132000. In contrast to this protein ceruloplasmin has been reported to contain two subunits of molecular weight 70000-80000. This was the reason for performing a direct comparison of polypeptide-chains from ceruloplasmin from difference mammalian sera. Ceruloplasmin was prepared from sera of man, pig, horse and rabbit. The reduced and alkylated proteins were gel filtered on a calibrated column of 6% agarose in 6M guanidine hydrochloride. The four proteins all contained a single main component cluting in the same position within errors of measurements. It is concluded that the four proteins investigated all contain a single polypeptide chain of very similar size. [ABSTRACT FROM AUTHOR]

After reduction and carboxymethylation, crystallized cytochrome b2 has been separated by gel filtration into two main fractions and a minor one The latter is probably an aggregate The apparent molecular weight of the major fractions has been determined by acrylamide gel electrophoresis m the presence of 0.1% sodium dodecyl sulfate, and found to be of the order of 36000 and 21000. Their amino acid composition is very different. Their sum gives a composition in good agreement with the composition corresponding to the minimum molecular weight of unfractionated cytochrome b2. On the basis of the evidence presented in the paper and of the known molecular weight of crystallized cytochrome b2, it is suggested that the protein is an oligomer of the type (αβ)4. [ABSTRACT FROM AUTHOR]

The Cbl-1 mutant of Bacillus subtilis 168 WT is the result of a spontaneous mutation presenting a pleiotropic character. In this paper, the results of the morphological and chemical analyses of the cell walls isolated from both strains are compared. When either the whole cells or the isolated cell walls of both strains were fixed with osmium tetroxide, the examination by electron microscopy showed extensive morphological differences in structure due to the partial destruction of the mutant cell walls. The chemical investigations performed on the isolated cell walls revealed surprising differences in chemical structure. In the cell walls of the mutant, teichoic acid was completely missing and was replaced by protein. A major protein-containing fraction accounting for about 50% of the material solubilized by autolysis of the mutant cell walls was isolated. Polyacrylamide gel electrophoresis showed that this fraction was composed mainly of a single protein of high molecular weight When denatured by sodium dodecyl sulfate treatment in the presence of β-mercaptoethanol, the protein yielded by gel electrophoresis two major bands with molecular weights of approximately 105000 and 155000, respectively. The molecular weight of the Cbl-1 cell wall protein determined by high speed sedimentation equilibrium was 255 600. [ABSTRACT FROM AUTHOR]

The effect of trypsin treatment with respect to the allosteric properties of yeast phosphofructokinase as well as its molecular weight has been studied. In the presence of fructose 6-phosphate trypsin desensitizes yeast phosphofructokinase to ATP inhibition. Under the same conditions, activation of this enzyme by the positive allosteric effector AMP, however, remains uneffected. Addition of MgATP instead of fructose 6-phosphate to the incubation medium containing trypsin protects the enzyme against ATP-desensitization. After prolonged incubation of yeast phosphofructokinase with trypsin the enzyme becomes inactivated both with fructose-6-phosphate and with MgATP. Using density gradient centrifugation sucrose media the effects of trypsin treatment on the sedimentation behaviour of yeast phosphofructokinase has also been investigated. With fructose 6-phosphate, trypsin converts the 560000 form of yeast phosphofructokinase into two enzymatically active forms of 510000 and 350000 daltons respectively, which are both e to ATP inhibition. On the other hand, in the presence of MgATP the peak of enzymatic activity sediments with 160000 daltons. This molecular form has the same high sensitivity to ATP inhibition as the untreated enzyme of molecular weight 560000. After the addition of fructose 6-phosphate, the 160000-molecule seems to dimerize giving an ATP-sensitive form with a molecular weight of 340000. In the light of studies about the existence of several interconvertible forms of yeast phosphofructokinase published recently by our laboratory, the results described in this paper are discussed in terms of the existence of dimeric and trimeric states, regarding the 16000-moiety as the trypsin modified monomer of the enzyme. Kinetic and allosteric properties of these states including conditions for their formation and stabilization are summarized and the peculiarities of the trypsin-unmodified and trypsin-modified enzyme are compared. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

1. Methionyl-tRNA synthetase from Escherichia coil extracts conserved at 0° has an apparent molecular weight of 175,000 estimated by gel filtration through Sephadex G-200, and a sedimentation coefficient of 6.5 S determined by sucrose density gradient centrifugation. 2. Incubation of the extract at 37° results in a time-dependent modification of methionyl-tRNA synthetase into an enzymatically active form, which has an apparent molecular weight of 54,000 and a sedimentation coefficient of 3.7 S. Under the same conditions of incubation, the Sephadex G-200 elution profile of several other aminoacyl-tRNA synthetases remains unaltered. 3. This modification of methionyl-tRNA synthetase is observed upon incubation of extracts derived from E. coli K12 or B cells grown under a variety of conditions (in minimal or enriched medium, at exponential or stationary phase). 4. The modified enzyme, which was partially purified by chromatography on hydroxylapatite, catalyses the formation of aminoacyl adenylate and also of aminoacyl-tRNA, using tRNAF as well as tRNAM. Furthermore, the Km values for various substrates of the two reactions do not differ significantly from those of the native enzyme. However, conversion into the modified enzyme is accompanied by a decrease in specific activity, and by an increase in the ratio of ATP-PP1 exchange capacity to aminoacyl-tRNA forming capacity. 5. Incubation of the modified enzyme at 37° in the presence of the substrates for each of the two reactions, followed by sucrose density gradient centrifugation in the presence of these substrates, does not lead to reversion to the native form. Thus, the ability to catalyze both reactions appears to be an intrinsic property of the modified enzyme. 6. In contrast to the behaviour of methionyl-tRNA synthetase in crude extracts, the partially or totally purified enzyme remains unaltered upon incubation at 37°. This observation has led to the demonstration that the extract contains a macromolecular component, readily separable from methionyl-tRNA synthetase, which is responsible for the conversion of the native to the modified enzyme. 7. Earlier studies on the purified enzyme [1] have shown that methionyl-tRNA synthetase from E. coli, with a molecular weight of 173,000, undergoes dissociation in the presence of 8 M urea or 6 M guanidine into four subunits which have a molecular weight of 43,000, and which appear to be homogenous by sedimentation analysis and polyacrylamide gel electrophoresis. In the light of those observations, the modification of methionyl-RNA synthetase reported in this paper is tentatively interpreted as the conversion, mediated by a macromolecular component in the extract, of the native enzyme into enzymatically active subunits. Work is in progress to elucidate the structural relationship between these enzymaticaily active subunits, and the subunits produced upon treatment of the purified enzyme with dissociating reagents. [ABSTRACT FROM AUTHOR]

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of α/β-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-14C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radio- activity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluoroehalcone- oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-14C]choleslerol-5α,6α-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions at those under which an intermediate was shown for both microsomal and soluble epexide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases. [ABSTRACT FROM AUTHOR]

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. tin a publication from the Cold Spring Harbor Laboratory, New York) for naming new proteinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KCI washes), the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 M NaCI/0.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25000–30000. [ABSTRACT FROM AUTHOR]

The properties of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells were determined. The enzyme has a molecular weight of approximately 40000. Its pH optimum is at pH 4.0, it is not inhibited by ethylenediaminetetraacetate (EDTA), and it is active at temperatures up to 60 °C. The enzyme cleaves adenovirus DNA in a stepwise manner. The limit digestion product has a molecular weight of 120000–200000. There is evidence that the cleavage reaction proceeds via an initial single-strand nick. Under the conditions tested the endonuclease did not seem to reveal a high degree of specificity as to the recognition of cleavage sites, or else the sites recognized occurred very frequently. [ABSTRACT FROM AUTHOR]

Amylomaltase, the inducible 4-α-glucanotransferase of Escherichia coli strain ML, has been purified to homogeneity. Its specific activity with a commercial maltose substrate was 500 mkat/kg protein (30 μmol glucose formed rain-1 mg protein-1). The purified enzyme, dependent on buffer concentration, exists in interconvertible low-molecular-weight (apparent molecular weight 71000) and high-molecular-weight (apparent molecular weight 370000) forms. The specificity of amylomaltase has been redefined. Hitherto, the enzyme was thought to be a glucosyltransferase, catalysing the transfer of single glucosyl units, and maltose has been regarded as its most important substrate. Amylomaltase is now shown to exhibit both glucosyl-transfer and 4-α-glucanosyl-transfer specificity. 4-α-Glucanosyl chains containing up to at least nine glucosyl units can be transferred. However, it is concluded that the transfer reaction by which amylomaltase action was originally expressed, does not take place, i.e., Maltose + maltose [This symbol cannot be presented in ASCII format] Maltotriose + glucose and that maltose has a restricted role as a substrate. This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate. It is confirmed that when a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing-end-group. Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release. The lag phase is overcome by addition of ‘priming’ (catalytic) concentrations of maltotriose or higher maltodextrins. An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose. The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous 1.4-α-glucans by E. coli. [ABSTRACT FROM AUTHOR]

A low molecular weight product (Mr 7000) synthetized on rat liver mitoribosomes is selectively extracted by chloroform/methanol (&frac21;). This products characterized by an unusual hydrophobicity, as reflected by its chromatographic behaviour, its high content in hydrophobic amino acids and its strong capacity to bind lipids. Bound lipids contain a high percentage of linoleic acid. [ABSTRACT FROM AUTHOR]

1. The molecular weight of 3-carboxy-cis-cis-muconate cyclase obtained from Aspergillus niger has been determined under dissociating conditions. Equilibrium sedimentation studies in the presence of 5 M guanidine hydrochloride (0.1% v/v with respect to 2-mercaptoethanol) and dodecylsulphate-calibrated polyacrylamide gel electrophoresis suggested a minimum covalent molecular weight of approximately 24000 for the reduced enzyme. 2. Sedimentation equilibrium studies in the presence of a denaturant but in the absence of a reducing agent gave a value of 47000 for the molecular weight. 3. The amino acid and carbohydrate composition of the enzyme was determined and the presence of one thiol per subunit was shown to be available for titration with 5,5'-dithio-bis(2-nitrobenzoic acid). 4. The enzyme was digested with trypsin, and peptide-mapping experiments confirmed the hypothesis that 3-carboxy-cis-cis-muconate cyclase consists of eight subunits of approximately 24000 molecular weight and identical chemical composition. [ABSTRACT FROM AUTHOR]

The amino-acid composition of a polypeptide fraction that contains a few closely related polypeptides was determined. These polypeptides are obtained in high yield from red kidney beans and stimulate RNA synthesis in a bacterial system and in mouse spleen lymphocytes. Peptide maps of the carboxymethylated fraction were prepared and tryptic peptides purified and analysed for total composition and amino acid sequence. The results revealed that the differences between the polypeptides are due to a few microheterogeneities in the form of amino acid exchanges, but no evidence for the existence of polypeptides of different size or overall structure were obtained. It is concluded that the purification method yields different types of highly similar polypeptides with a molecular weight in the range of 10000, corresponding to about 80 residues, and an unusually high cysteine content of 19 mol/100 mol. These facts show that the present polypeptides are different from all previously analysed factors stimulating RNA synthesis. [ABSTRACT FROM AUTHOR]

When cultures of biotin-requiring mutant of Brevibacterium divaricatum NRRL-2311 were treated in early logarithmic phase with penicillin (2–3 units/ml) in a glucose-mineral medium under excess supply of biotin, large amounts of a high-molecular-weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of 1-h and 24-h penicillin-treated cultures, showed that the main constituents were peptidoglycan components of noncross-linked structure bearing both L- and D-alanine residues: evidence was also obtained for the occurrence of extractable lipid material, non-amino sugars and organic phosphate. Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin-requiring mutant of M. glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricatum strain. This suggests that excretion of peptidoglycan material by the two biotin-requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross-link formation in peptidoglycan induced by penicillin. Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin-treated Brev. mutant. Suitable labelling experiments with L-[U-14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion. [ABSTRACT FROM AUTHOR]

Creatine phosphokinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is the major constituent of the ‘low-salt-soluble’ proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti-(electric organ creatine phosphokinase) antibodies are specific for the muscle-type enzyme and do not cross-react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine-receptor-enriched membranes show that this enzyme is as the ‘low-salt-soluble’ electric organ enzyme of the muscle-specific type. In vitro translation of electric organ poly(A)-rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain-type polypeptides are synthesized. The subunits of the brain-specific enzyme differ in molecular mass (Mr &assymp; 42 000) and isoelectric properties (pl &assymp; 7.0–7.2). The unexpected finding that the brain forms are more basic than the muscle-specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion-exchange chromatogaphy properties. [ABSTRACT FROM AUTHOR]

Three dimeric glutamyl-tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts. 1. The chloroplast enzyme (Mr = 110000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity. 2. Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GIuRS E, has eukaryotic (wheat germ) tRHA specificity. Both enzymes exhibit a molecular weight close to 160000. 3. Each of these enzymes co-eluate on hydroxyapatite and phosphocellulose chromatographies with an unstab|e active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers. [ABSTRACT FROM AUTHOR]

Two 1,3; 1,4-β-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3; 1,4-β-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants. [ABSTRACT FROM AUTHOR]

Describes the purification of a bovine brain thyrotropin-releasing-factor deamidase to apparent homogeneity. Molecular weight; Gel filtration; Sodium dodecylsulfate gel electrophoresis; Peptide bonds; Inhibitors of thyroliberin deamidation.

The stability of the native quaternary structure of bovine α-crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7–11), ionic strength (0.01–0.5), temperature (6–60 °C) and calcium ion concentration (0 and 10 mM). Three successive transitions are distinguished at 20 °C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of ‘alkali-modified α-crystallin’, which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified a-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3. Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 °C with formation of ‘temperature-modified a-crystallin’. which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37 °C a reversal of this transition or aggregation is indicated. These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of α-crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure. [ABSTRACT FROM AUTHOR]

The N-terminal tryptic peptide of Crithidia oncopelti cytochrome c557 X-Pro-Me3Lys-Ala-Arg in which X represents an unknown N-terminal blocking group was characterized by electrophoresis at pH 2 and by 1H and 13C nuclear magnetic resonance.1H-NMR spectra of the tryptic peptide suggested that the blocking group X was N,N-dimethylproline although the electrophoretic mobility of the peptide suggested a larger molecular weight. The peptides X-Pro-Me3Lys and X-Pro were generated by treatment of the tryptic peptide with thermolysin and carboxypeptidase and the free blocking group X was prepared by acid hydrolysis. Comparison of the 1H-NMR spectra of these peptides with spectra of synthetic N,N-dimethylproline and N,N-dimethylprolylproline demonstrated that the blocking group was indeed N,N-dimethylproline. The 13C-NMR spectrum of the tryptic peptide was consistent with this conclusion although unambiguous assignments to all resonances could not be obtained because of the small amount of material available. The origin of the dimethylproline blocking group is discussed. [ABSTRACT FROM AUTHOR]

DNA methyltransferase was purified 310-fold from a green alga, Chlamydomonas reinhardi vegetative cells. The native enzyme of molecular weight 55 000–58 000 catalyzed the transfer of methyl groups from S-adenosylmethionine to the 5 position of cytosine in DNA. Native DNA accepted methyl groups 10-fold more than did denatured DNA. The sequence specificity analysis of methylated deoxycytidine in vitro revealed that the enzyme introduces methyl groups preferentially into sequences containing 5′d(T-mC-R)3′. Kinetic analysis of the reaction indicated that the enzyme obeys a random sequential mechanism. The extent of saturation with methyl groups depends upon the species from which the DNA was obtained. Kinetic analysis of the reaction catalyzed by RNA polymerase II has indicated that DNA methylation decreases the rate of initiation of RNA synthesis, but does not affect the rate of RNA chain elongation. [ABSTRACT FROM AUTHOR]

Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocysteamine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km value for S-methyladenosylhomocysteamine was 0.6 μM and about 60 μM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5′-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (K1 value of about 0.3 μM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 nM). Spermine synthase had no requirements for metal or other cofactors. [ABSTRACT FROM AUTHOR]

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55000. Conditions of optimal sodium chloride concentration and pH at 25°C were 0.25–0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]-sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-3,6-anhydro-α-D-galactopyranosyl-(1→3)-D-galactose 4-O-sulphate]. [ABSTRACT FROM AUTHOR]

From the cell membranes of the sponge Geodia cydonium a component was isolated and purified which inhibits the aggregation factor isolated from the same source; the component was termed anti-aggregation receptor. This molecule was characterized as a glycoprotein (54 % neutral carbohydrate) and its molecular weight is in the range of 180000. One biological site of the anti-aggregation receptor was determined to be D-galactose. Indirect evidence presented seems to indicate that this molecule is present in an active form in aggregation-deficient cells and absent in aggregation-susceptible cells. [ABSTRACT FROM AUTHOR]

Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen-enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7–11 U/mg protein, or 4–5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13500cal/mol (56.5 kJ/mol), corresponding to a Q10 of 2.2. In the presence of glucose 6-phosphate or Na2SO4, the enzyme showed a broad pH optimum between pH 6.8-9.2. In the absence of these ligands and in particularly in the presence of Mg2+, the enzyme is sensitive to small changes of pH in the interval pH 7.4- 8.4. During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing. During 24-h incubations, synthase I undergoes a spontaneous, temperature-dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6-phosphate, Na2SO4, inorganic phosphate, UDP and glycogen. Mg2+ and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years. [ABSTRACT FROM AUTHOR]

β-Actinin was purified to homogeneity from water extracts of chicken leg muscle by chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. 60-100 mg of protein was obtained from 1 kg of muscle. The molecular weight estimated from gel electrophoresis in the presence of sodium dodecylsulfate was 65 000, from gel filtration on Sephadex G-200 66 000, and from sedimentation equilibrium experiments 67 000. The results show that β-actinin is a monomeric protein. The amino acid composition was determined and differed from β-actinin of rabbit muscle. Double-immunodiffusion experiments in Ouchterlony plates using anti-chicken β-actinin indicated that an immunologically indistinguishable form of this protein is present in muscle and nonmuscle tissues of the chicken. The distribution of β-actinin is therefore similar to G-actin and parvalbumin-like protein. β-Actinin was found to accumulate in differentiating primary muscle cell cultures after 120 h approximately together with myosin and actin, proteins necessary for myofibril assembly and functioning. Experiments to investigate the localization of β-actinin, using the indirect immunofluorescence technique, showed a regular, intensive cross-striation pattern within the I band of isolated myofibrils. An electronmicroscopic investigation demonstrated that β-actinin does not polymerize to filamentous structures under conditions where G-actin is transformed to F-actin. It is able to inhibit the polymerization of G-actin and promotes the depolymerization of preformed F-actin filaments in vitro. The immunological, physico-chemical and electronmicroscopic data show that β-actinin is a quite distinct protein from actin. [ABSTRACT FROM AUTHOR]

A DNA-stimulated ATP-γ-phosphohydrolase of molecular weight 75 000 was purified from Escherichia coli cells. The ATPase, a globular molecule (identical probably with an ATPase described previously by Richet and Kohiyama in 1976) shows specificity for adenine nucleotides, it prefers single-stranded DNA as the cofactor, it exhibits a complicated mode of response to variations of the cofactor concentration and it is devoid of nuclease activity. Preparations derived from rep3 mutant cells yield widely varying amounts of an apparently normal ATPase. [ABSTRACT FROM AUTHOR]

Mung bean nucleotide pyrophosphatase isolated in a crystalline and homogeneous form as a dimer with a molecular weight of 65000 was converted by AMP into a tetramer. The tetramer was enzymatically active with altered kinetic properties. This conversion of the dimeric form by AMP to a tetrameric one was prevented by treating the dimer with p-hydroxymercuribenzoate. The molecular weight of the p-hydroxymercuribenzoate-treated enzyme was determined to be 32700 by a combination of Stokes' radius (2.4 nm) and sedimentation velocity (s20, w = 1.9 S), by thin-layer gel chromatography on superfine Sephadex G-200 and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The monomer obtained by treatment of the native enzyme with p-hydroxymercuribenzoate was isolated by passage of the dissociated enzyme through a column of Biogel P-200. The monomer was optimally active at 37 °C, whereas the dimer and tetramer were active at 49 °C. All the three enzyme forms were maximally active at pH 9.4. The Km and V (measured as rate of FAD hydrolysis per mg protein) for FAD of the three enzyme forms were for the monomer, 0.5 mM and 7.0 µmol min-1, for the dimer, 0.25 mM and 3.3 µmol min-1 and for the tetramer, 0.58 mM and 2.5 µmol min-1, respectively. The time course of the reaction of the monomer was linear and comparable to the initial fast rate of the dimer. The monomer was not converted to a tetramer or a dimer on the addition of AMP; and it was irreversibly inhibited by urea and EDTA. ATP and ADP were noncompetitive inhibitors of the monomer. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

Ultraviolet light produces pyrimidine dimers in cellular DNA. In mouse cell lines the great majority of these pyrimidine dimers are not excised, but remain in high molecular weight DNA. Hence the DNA used as template for DNA synthesis in the first generation after ultraviolet irradiation must contain such dimers. In two mouse cell lines (L5178Y and 3T3), alkaline sucrose sedimentation studies showed that the DNA pulse-labelled shortly after irradiation contained gaps, which by analogy with the situation in Escherichia coli, are presumed to be opposite the pyrimidine dimers. In contrast such gaps could not be detected in DNA pulse-labelled several hours after irradiation, despite the fact that dimers must have been present on the template strands. A possible explanation is that gaps were still being formed, but were filled in rapidly and were therefore not detected. This hypothesis was tested as follows. Either 1 or 8 h after ultraviolet irradiation cells were pulse-labelled in the presence of theophylline. This purine inhibits the filling in of gaps presumably by binding to the DNA in the gap. Even in the presence of theophylline, the late-synthesized DNA was considerably larger than the early-synthesized DNA. This suggests that at late times after irradiation, gaps were either not formed at all opposite the dimers, or were so transient that theophylline could not bind and inhibit their filling in. A model for synthesis of DNA on templates containing pyrimidine dimers in mammalian cells, based on these findings and other recent results, is discussed. [ABSTRACT FROM AUTHOR]

Aspartokinase I-homoserine dehydrogenase I from Escherichia coli K 12 was subjected to mild proteolysis. A fragment carrying only the desensitized homoserine dehydrogenase activity was purified. It is a dimer having a subunit molecular weight of 55 000 as opposed to 86 000 for the subunit of the native tetrameric enzyme. On the other hand, a threonine sensitive aspartokinase devoid of homoserine dehydrogenase activity was extracted from the mutant Gif 108. The purified protein is shown to have subunits shorter than those of the wild-type enzyme (molecular weight 47 000). It was shown that the two catalytic activities of aspartokinase I-homoserine dehydrogenase I are sequentially distributed on the single polypeptide chain: the aspartokinase activity is located in the amino-terminal section and the homoserine dehydrogenase in the carboxyl-terminal section. The results are discussed in relation to the configuration of the native enzyme and with the possible origin of the bifunctional protein. [ABSTRACT FROM AUTHOR]

A study of the structural requirements for activity of low molecular weight compounds in the donor or peptidyl site of ribosomal peptidyl transferase requires methods for the chemical synthesis of potential donor substrates. Methods are described here for the preparation of cytidylyl. (3' → 5')-cytidylyl-(3' → 5')-2'(3')-O-(N-[³H]acetyl-L-leucyl)-adenosine and cytidylyl-(3' → 5')- 2'(3')-O-(N-[³H]acetyl-L-leucyl)-adenoaine and the corresponding L-phenylalanine derivatives. A major difficulty overcome was the severe restriction placed on possible synthetic routes by the extreme alkali lability of the aminoacyl linkage. The synthetic route developed involved the stepwise synthesis of CpCpA and CpA appropriately protected with acid-labile protecting groups. The free terminal 2'(3')-hydroxyls were aminoacylated, all protecting groups removed and the α-amino groups acylated with [³H]acetic anhydride. The chemically prepared N-acetyl-aminoacyl-trinucleotide fragments had similar activity in the ribosome-catalysed fragment reaction as reported for the corresponding pentanucleotide fragments obtained from biological sourecec. Further, the inactivity of the N-acetyl-aminoacyl-dinucleotide fragments was confirmed under a wider range of conditions than those previously described. [ABSTRACT FROM AUTHOR]

Mitochondrial ATPase from rat liver has been highly purified to a maximum specific activity of 110 in Tris-bicarbonate buffer at pH 8.0. Sedimentation velocity studies in the analytical ultracentrifuge show that the enzyme sediments as a single symmetrical peak with a sedimentation coefficient (sº20, w, w) of 12.9 S. Molecular weight determinations by both high-speed sedimentation equilibrium and polyacrylamide gel chromatography indicate a molecular weight of 360 000 ± 10 000. Polyacrylamide gel electrophoresis of the enzyme dissociated by sodium dodecyl sulfate reveals one major and three minor bands with molecular weights of 53 000, 28 000, 12 500, and 8000-9000. A calculated molecular weight of approximately 365 000 is obtained by assuming the presence of six subunits of molecular weight 53 000 and one of each of the lighter subunits. Like the beef-heart enzyme, rat-liver ATPase activity is lost on incubation at 5 °C and examination by high-speed sedimentation equilibrium at 5 °C indicates that the molecule is extensively dissociated to components of low molecular weight. The enzymatic activities of both rat-liver-mitochondrial and beef-heart-mitochondrial ATPases are sensitive to the anion of the buffer used at pH 8.0 with activity decreasing in the following series: HCO3- > maleate > chloride > acetate = sulfate. Sulfite, chromate and dinitrophenol stimulate the activity of either enzyme in Tris-sulfate buffer, but only sulfate increases the activity seen in Tris-bicarbonate buffer. Aurovertin decreases the enzymatic activity to approximately the same level, regardless of the anion(s) present. The enzyme is insensitive to oligomycin. [ABSTRACT FROM AUTHOR]

The action patterns of liver and muscle glycogen synthetases and of muscle phosphorylase b on glycogen samples of different molecular weight and on β-amylase limit dextrins were studied. For this purpose a method for measuring the number of newly added glucose residues that are at non-reducing ends was developed. It was found that glucose transfer to the non-reducing ends of glycogen catalyzed by liver glycogen synthetase and muscle phosphorylase followed a Poisson distribution. The number of outer chains in the glycogen molecules available to both enzymes appeared to be smaller than the actual number of outer chains in the polysaccharide. The number of such available chains diminished as the glycogen was heavier. For the same glycogen sample, the number of available chains to phosphorylase appeared to be equal or smaller than that, to glycogen synthetase. It was found that muscle phosphorylase transferred 1.2 to 1.4 glucose moieties successively per outer chain, independently of the molecular weight, of the glycogen. The number of glucose units added successively to the non-reducing ends of glycogen by liver glycogen synthetase increased from 1.7 to 6.8 with the molecular weight of the polysaccharide. Both phosphorylase and liver glycogen synthetase transferred more glucose units in a repetitive way to the same outer chain of the β-amylase limit dextrin than to the same outer chain of the undegraded glycogen. Muscle glycogen synthetase transferred a greater number of glucose units successively per outer chain of glycogen than the liver enzyme. [ABSTRACT FROM AUTHOR]