Determination of the size of polyphosphates with polyphosphate glucokinase

A procedure for determining the size of inorganic polyphosphates of chain lengths up to about 750 is described. It involves reducing the size with polyphosphate glucokinase to a chain length that can be accurately determined by polyacrylamide gel electrophoresis. This measurement along with determination of the glucose-6-P formed and the total phosphate of the original polyphosphate permits calculation of the chain length. The accuracy of this method has been demonstrated by comparison with other reliable procedures. Thus far, it is the only method available for sizing long chain polyphosphates with nmol quantities.     

Polyphosphate glucokinase from Propionibacterium shermanii. Kinetics and demonstration that the mechanism involves both processive and nonprocessive type reactions

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Observation of residual dipolar couplings in short peptides

Residual dipolar couplings provide information on the orientation of individual bond vectors with respect to a unique set of molecular axes. We report that short peptides from 2 to 15 amino acids in length of arbitrary sequence exhibit a modest range of residual dipolar couplings when aligned in either strained polyacrylamide gels or alkyl-PEG bicelles. The absence of significant line broadening in gels suggests peptides align predominantly through steric interactions with the polyacrylamide matrix. However, broadening of NMR lines for a subset of residues aligned in bicelles indicates some peptides bind weakly to these lipid disks, yet a weak negative correlation between the couplings measured in gels and bicelles is consistent with steric hindrance playing a role in both media. The observation of dipolar couplings for peptides of length 10-15 suggests the statistical segment lengths of polypeptide chains must often be >10-15 residues, with data from denatured proteins indicating even larger values. Presumably, local side-chain backbone interactions severely restrict chain flexibility, with the cumulative effect of many such restrictions giving rise to biases in chain direction that may persist for the entire length of a protein chain. Comparison of experimental dipolar couplings for peptides with couplings calculated for ensembles of conformations generated by molecular dynamics should permit evaluation of the accuracy of molecular mechanics potentials in reproducing sequence-specific preferences for phi and psi angles.     

Determination of poly(ADP-ribose) chain length distribution on polyacrylamide gels by silver staining.

Silver staining and polyacrylamide gel electrophoresis were used to visualize chain length distribution of poly(ADP-ribose) enzymatically synthesized from NAD by rat liver nuclei. The method described has the advantage that synthesis does not require radioactive-labeled NAD, and microgram quantities (greater than 5 micrograms) of poly(ADP-ribose) can be resolved and visualized as discrete bands according to chain lengths which range from 8 to 60 residues. This method can be applied to estimate size distribution of poly(ADP-ribose) chains in cells or tissues.     

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.     

Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures.

Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.     

The mechanism of utilization of polyphosphate by polyphosphate glucokinase from Propionibacterium shermanii

The phosphorylation of glucose by polyphosphate glucokinase with both long- and short-chain polyphosphates has been shown to occur by either a nonprocessive mechanism, i.e. with repeated association and dissociation of the polyphosphate from the enzyme after each phosphorylation or by a quasiprocessive mechanism in which several phosphorylations occur prior to the release of polyphosphate and the reassociation with the enzyme. In contrast, the phosphorylation of ADP to ATP by polyphosphate kinase is by a strictly processive mechanism; the phosphorylation occurs without release of the polymer from the enzyme prior to termination of the reaction (Robinson, N. A., Clark, J. E., and Wood, H. G. (1987) J. Biol. Chem. 262, 5216-5222). The demonstration that the mechanism is quasi-or nonprocessive was accomplished by electrophoresis using a variety of concentrations of polyacrylamide gels which made it possible to detect the intermediate sizes formed during the reactions. It also has been shown that all chains longer than about 100 are used simultaneously, but with chains of less than 100 residues, there is preferential utilization of the longest chains. Thus a narrow range of sizes is formed from a heterogeneous mixture of long chains. It is this formation of the narrow range of sizes that makes it possible to use polyphosphate glucokinase for the determination of the average size of long chains (Pepin, C. A., Wood, H. G., and Robinson, N. A. (1986) Biochem. Int. 12, 111-123).     

Polymer length of teichuronic acid released from cell walls of Micrococcus luteus.

Teichuronic acid released from its phosphodiester linkage to peptidoglycan in the cell walls of Micrococcus luteus by mild acid treatment is resolved into a ladderlike series of bands by electrophoresis on polyacrylamide gels in the presence of borate. Each band of the ladder differs from its nearest neighbor by one disaccharide repeat unit, ----4)-2-acetamido-2-deoxy-beta-D-mannopyranuronosyl-(1----6)- alpha-D-glucopyranosyl-(1-. Acid-fragmented teichuronic acid, after conversion to the phenylamine derivative, was fractionated by preparative-scale molecular sieve column chromatography, which produced a series of elution peaks. Fast-atom-bombardment mass spectrometry of the smallest member of the series determined its molecular weight and established its identity as the phenylamine derivative of one disaccharide repeat unit of teichuronic acid. Homologous fractions of the same series were used to index the ladder of bands obtained by polyacrylamide gel electrophoresis from samples containing a more extensive distribution of polymer lengths. Nearly native teichuronic acid consists of polymers with a broad range of molecular sizes ranging from 20 to 55 disaccharide units. The most abundant species are those which have 25 to 40 repeat units. Prolonged treatment of teichuronic acid with the acid conditions used to release it from peptidoglycan causes gradual fragmentation of the teichuronic acid.     

A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

A rapid and effecient method for the isolation of proteins from polyacrylamide cells.

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.     

Isolation of messenger RNA coding for eggshell protein of the DNA-eliminating nematode Ascaris lumbricoides

Poly(A)-containing RNA from polyploid uterine epithelial cells of Ascaris lumbricoides has been isolated by poly(U)-Sepharose chromatography. The bulk of poly(A)-containing RNA migrates as 18-S RNA in formamide/polyacrylamide gels. In a cell-free wheat germ system, this RNA directs the synthesis of a polypeptide with identical migration behavior in dodecylsulphate/urea/polyacrylamide gels as the polypeptide isolated from the proteinaceous eggshell. The two proteins reveal almost identical peptide patterns in fingerprint analysis. The authentic eggshell protein has been identified as a glycoprotein with a molecular weight of about 10000, as determined by dodecylsulphate/polyacrylamide gel electrophoresis. The apparent discrepancy between mRNA length and the required coding length for the protein is discussed.     

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that the synthesis and utilization of polyphosphate is by a processive mechanism

The mechanism of synthesis of inorganic polyphosphate by polyphosphate kinase (EC 2.7.4.1) from Propionibacterium shermanii is shown to be processive. Analysis of the synthesized polyphosphate on polyacrylamide gels, which resolve on the basis of molecular weight, proves that the elongation reaction occurs without dissociation of intermediate sizes of the polymer from the enzyme. As a consequence, only high molecular weight polyphosphates are synthesized. The mechanism is processive both in the presence and absence of basic protein. It has been shown previously that basic proteins stimulate the synthesis of polyphosphate (Robinson, N.A., Goss, N.H., and Wood, H.G. (1984) Biochem. Int. 8, 757-769). In addition, using a similar method, it is shown that the reverse reaction, the utilization of polyphosphate to phosphorylate ADP, occurs by a processive mechanism. Accordingly, polyphosphates formed by polyphosphate kinase in the cell would be entirely high molecular weight.     

Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA.

A protein similar to that previously demonstrated on poliovirus RNA and replicative intermediate RNA (VPg) was found on all sizes of nascent viral RNA molecules and on the polyuridylic acid isolated from negative-strand RNA. 32P-labeled nascent chains were released from their template RNA and fractionated by exclusion chromatography on agarose. Fingerprint analysis using two-dimensional polyacrylamide gels of RNase T1 oligonucleotides derived from nascent chains of different lengths showed that a size fractionation of nascent chains was achieved. VPg was recovered from nascent chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5' terminus could be demonstrated on nascent RNA, and the yield of VPg was consistent with one molecule of the protein on each nascent chain. These results are consistent with the concept that the protein is added to the 5' end of the growing RNA chains at a very early stage, possibly as a primer of RNA synthesis. Analysis of the polyuridylic acid tract isolated from the replicative intermediate and double-stranded RNAs indicated that a protein of the same size as that found on the nascent chains and virion RNA is also linked to the negative-strand RNAs. It is likely that a similar mechanism is responsible for initiation of synthesis of both plus- and minus-strand RNAs.     

The instantaneous monitoring of polyacrylamide gels during electrophoresis.

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.     

The coordinate synthesis and cotranslational assembly of type I procollagen

Nascent polysome-associated type I procollagen pro-alpha-chains isolated from chick embryo tendon fibroblasts were examined for their proteinase resistance. The distribution of chain sizes and their proteinase resistance were also determined following chain elongation in an in vitro readout system in the absence of chain initiation factors. Chains were labeled with [14C]proline in the cells and with [3H]proline in the readout system. Differences in the ratios of 14C to 3H in the double-labeled nascent chains before and after chymotryptic digestion, determined by slicing and counting polyacrylamide gels after electrophoresis, permitted analysis of the relative stabilities of in vivo and in vitro elongated portions of the chains. In confirmation of earlier work, the polysome-bound nascent procollagen contained chymotrypsin, chymotrypsin plus trypsin, and pepsin-resistant alpha-chain size components. The readout system data showed that the full length chains produced in the cell were more resistant to digestion than the fully elongated readout-completed chains. The protease resistance of the chains was taken to indicate the registration of the chains prior to the induction of helix formation during the isolation procedure. These data support the model in which chain selection and folding are facilitated by the organization of the attachment of the ribosomes to the endoplasmic reticulum surface.     

Electrophoresis of DNA in ultrathin planar-format linear polyacrylamide

Linear or un-cross-linked polyacrylamides have been employed successfully in the field of capillary electrophoresis for the separation of nucleic acids. Typical acrylamide concentrations for those applications range from 3% to 14% (wt/vol), with consistencies ranging from virtually liquid to moderately viscous. Due to the absence of cross-links, and the relatively fluid nature of linear polyacrylamide at typically employed concentrations, its use in planar (slab) gel electrophoresis has been overlooked. We describe herein the application of ultrathin (100 μm) high-viscosity slabs of linear polyacrylamide to planar electrophoresis of nucleic acid fragments. The approach we describe is rapid and yields high-resolution separations of nucleic acid fragments in linear polyacrylamide supports. The mobilities of DNA fragments of various lengths in a range of concentrations of linear polymer are compared with those observed for conventional cross-linked gels. The reptative migration of larger DNA fragments in linear polymers is predictable from the models derived from work with cross-linked acrylamide and agarose. The migration of smaller fragments, however, is not entirely predicted by the Ogston model. The relative mobilities observed for very small DNA fragments are approximately half those predicted by the Ogston regimen.     

Separation of the DNA molecules beyond conventional size limits by gel electrophoresis with sodium dodecyl sulfate.

Electrophoretic mobility of DNA through polyacrylamide as well as agarose gels is greatly increased by sodium dodecyl sulfate (SDS). DNA molecules well beyond the conventionally separable size limits are separated readily and rapidly by gel electrophoresis with SDS in a conventional static electric field. Furthermore in optimal concentration gels DNA molecules of similar molecular sizes are separated better from one another in the presence of SDS than without it. Evidence is presented that SDS may act at least in part by altering conformation of DNA. This simple and readily available means for high resolution separation of hitherto impossible sizes of DNA molecules in polyacrylamide and agarose gels in an ordinary static electric field should find general use in molecular genetic analyses. Structural analyses of DNA-protein complexes are also facilitated by virtue of the simultaneous separation of the DNA and protein components on the same gel lane.     

The molecular weight of J chains derived from human immunoglobulin M

J chain was isolated from sulphonated human immunoglobulin M molecules by electrophoresis on polyacrylamide gels. When determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide gels, the molecular weight of the protein was about 27000. After suspension in 5m-guanidine hydrochloride solution for 21 days, two groups of three bands appeared on the gels. Most of the protein dissociated to components of molecular weight 15000. The molecular weight of purified J chain was also determined by ultracentrifugation. In borate-saline solution the average weight-average molecular weight was about 29000. The molecular weight slowly decreased upon prolonged exposure to guanidine hydrochloride, and after 14 days the minimum molecular weight was about 15000. Some association between chains still existed. These data suggest that J chain derived from the paraprotein exists in borate-saline solution as dimers held by strong non-covalent forces.