In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

Characterization of a cytoplasmic polyhedrosis virus from Estigmene acrea (Lepidoptera)

A cytoplasmic polyhedrosis virus (CPV) containing a segmented double-stranded RNA genome was isolated from Estigmene acrea larvae by isopycnic centrifugation in a sucrose density gradient. Ten double-stranded RNA segments with molecular weights (MW) from 2.8 to 0.67 × 10⁶ were separated by agarose gel electrophoresis. A total of ten virus proteins ranging from 14,000 to 128,000 MW were detected after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A MW of 28,500 was determined for E. acrea CPV occlusion body protein.     

Characterization of a soluble carnitine acetyltransferase from Candida tropicalis

Carnitine acetyltransferase was purified from the cytoplasmic fraction of Candida tropicalis grown on alkanes in continuous culture. By ion-exchange chromatography the enzyme was resolved in two fractions with the same specific activity of 80 U/mg. The molecular mass of both enzyme forms, determined by non-denaturing gradient gel electrophoresis, was 540 kDa. After SDS electrophoresis only one band of 64 kDa was detected indicating that both enzymes are oligomers each containing eight subunits. Isoelectric focusing in agarose under non-denaturing conditions demonstrated the presence of at least four different charged species in the pH range between 5.6 and 6.7. After isoelectric focusing in 9 M urea/1% Nonidet P-40 gels, both enzyme forms were resolved into four bands. Peptide mapping, performed by cyanogen bromide cleavage of polypeptides separated by denaturing isoelectric focusing followed by second-dimension SDS electrophoresis, revealed a very high degree of homology between these polypeptides. The presence of the octameric form of carnitine acetyltransferase already in the starting material was demonstrated by non-denaturing gradient gel electrophoresis and immunoblotting. Antibodies against carnitine acetyltransferase from C. tropicalis ATCC 32113 formed precipitation lines with extracts from several Candida species but not with extracts of Candida utilis, Candida ethanothermophilum and an another strain of C. tropicalis.     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Polyacrylamide gel electrophoresis of baculovirus proteins: A simplified and sensitive modification for differentiating between isolates

A rapid and sensitive modification of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of baculovirus structural proteins has been developed. Polyhedral inclusion bodies (PIBs) were pretreated with 1% SDS and 0.5% 2-mercaptoethanol (2-ME) for 30 min at pH 7.2, washed to remove soluble material, dissociated with Laemmli's sample buffer, and analyzed by SDS-PAGE. When the gels were stained with silver nitrate, as little as 48 μg of protein, comprising both polyhedrin and virion proteins, could be resolved on the same gel. Pretreatment with SDS and 2-ME eliminated the need to further purify PIBs by sucrose gradient centrifugation, since gel profiles of PIB proteins before and after such centrifugation were indistinguishable. The method was used to distinguish between the nuclear polyhedrosis viruses (NPVs) of the following species: Mamestra brassicae, Wiseana cervinata, Autographa californica, Mythimna convecta, Persectania dyscrita, Spodoptera exigua, S. frugiperda, Anthela varia, Pterolocera amplicornis, and Heliothis punctiger. Cross-transmission of A. californica NPV to H. punctiger and M. convecta and of M. convecta NPV to P. dyscrita was confirmed by analysis of progeny virus proteins.     

Absence of major differences between soluble proteins from haploid gametophytes and diploid sporophytes in the green alga Ulva mutabilis Føyn

Soluble proteins from haploid gametophytes and diploid sporophytes of the marine green alga Ulva mutabilis Føyn have been reexamined, using polyacrylamide gel electrophoresis and isoelectric focusing. A two-dimensional system resolved about 150 protein spots. In contrast to an earlier report (Hoxmark (1976) Planta 130, 327–332), no major differences could be detected between soluble proteins from the two generation types by any of the methods used.To whom correspondence should be addressed     

Comparison of protein patterns in cockroach muscles innervated by fast or slow neurons

The protein composition of six of the coxal depressor muscles of the leg of the cockroach, Periplaneta americana, was analyzed by one and two dimensional SDS—polyacrylamide gel electrophoresis in conjunction with a sensitive silver stain. The proteins in each muscle were fractionated according to their solubilities in either low salt buffer, 1%NP-40, or 2%SDS. The gel patterns resulting from the electrophoresis of each of the fractions from “fast”, “slow”, and “mixed” type muscle were compared with reference to the innervation of the muscle by a particular motor neuron. More than 50 proteins were detected which were not common to all three types of muscle. These proteins were found to be distributed fairly equally amongst each of the three fractions. The large number of differentially expressed proteins observed, are not likely to be involved in specific neuromuscular recognition, but instead are probably correlated with the physiological state of the muscle.     

Quantitation of specific proteins in polyacrylamide gels by the elution of fast green FCF

The quantitation of proteins in polyacrylamide gels stained with Fast Green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595–602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406–416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aquoeus pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method to several different proteins and our results indicate: (a) The quantity of Fast Green eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1–800 μg). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gel, (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.     

Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons. The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%. Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins. Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass. Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.     

Addition of proteins to the cylindrical gel embedding medium for transverse molecular-weight markers in two-dimensional gel electrophoresis

As an aid in the comparison of different complex mixtures of proteins resolved by two-dimensional electrophoresis, a simple method which results in the electrophoresis of molecular-weight standards as appropriately migrating, highly resolved bands extending across the entire second-dimension slab gel is described. The proteins to be used as markers are included in the molten agarose mixture used to affix the first-dimension cylindrical gel atop the second-dimension slab gel. As the proteins which are resolved in the first dimension migrate through the second-dimension slab gel, the marker proteins also migrate, experiencing the same electrophoretic conditions as the sample proteins in the immediate vicinity. If the same protein is resolved in the first dimension and also used as a marker, it electrophoreses in the second dimension as a spot intersected by a band traversing the entire gel. This sensitive method is applied to a comparison of soluble seed proteins of two cotton species, Gossypium hirsutum and G. arboreum, using G. hirsutum seed protein as the molecular-weight marker. Other applications are described.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Microscopic and Protein Expression Analysis of Grain Weight Variation within the Spike of Triticum aestivum

The kernels possess significant grain weight variation in one wheat (Triticum aestivum L) plant because of their different positions within the spike. In order to understand the molecular basis of weight, a proteomic approach, employing twodimensional electrophoresis and matrix-assisted laser desorptionfionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins between two kinds of kernels, the high weight kernel (large kernel) and the low weight kernel (small kernels) with different positions within spikes of one wheat cultivar, Shannong. Microscopic observation showed that the endosperm cells in large kernels enlarged their volume and accumulated storage materials at grain filling stage (17 days after anthesis, DAP), whilst those in small kernels were mainly in cell division with larger vacuoles during this period. Proteins were extracted from the kernels at this time, and resolved using 24-cm immobilized pH gradient strips with a pH 3–10 linear gradient in the first dimension and a 12.0% sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension. About 750 protein spots in each gel were resolved after electrophoresis and 45 proteins were expressed significantly differently between the two kernels. MALDI-TOF MS characterization of the resolved spots in the two samples enabled us to identify 28 proteins whose levels were altered; 19 and 9 proteins were up-regulated in high and low weight kernels, respectively. In particular, proteins beneficial to materials synthesis and transmission increased distinctly in high weight kernels, while in low weight kernels, proteins involved in cell division were increased. The kernels with different position in spike might be at different physiological status, and this might be one of the causes resulting in grain weight differences within one spike.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Changes of soluble protein populations during organogenesis of mouse embryos as revealed by protein mapping

Soluble proteins from whole mouse embryos of different stages of organogenesis and from embryonic and adult organs were investigated by employing the protein mapping technique combining isoelectric focusing with polyacrylamide gel electrophoresis or with SDS electrophoresis. Protein patterns composed of more than 400 spots were obtained. Considerable qualitative differences in the composition of these patterns were obvious at the developmental stages investigated. Phase-specific protein populations were found and the members of such populations had some properties in common. A population of proteins of relatively high molecular weight disappeared after Day 9 post conceptionem (p.c.). A large protein population arose between Days 9 and 14, not characterized by a certain range of molecular weights or isoelectric points, and is present in several, if not in most tissues of Day 14 embryos. A population of proteins typical of an organ or possibly organ-specific appeared later than Day 14 p.c., at the time when morphological differentiation of organs is actually completed.     

Spherosomes of Castor Bean Endosperm: MEMBRANE COMPONENTS, FORMATION, AND DEGRADATION

The membrane components of the castor bean spherosomes were characterized. The storage triacylglycerols of isolated spherosomes were extracted with diethyl ether, and the membrane was isolated by sucrose gradient centrifugation. It had an apparent equilibrium density of 1.12 grams per cubic centimeter, and possessed an antimycin A-insensitive NADH cytochrome c reductase and an acid lipase. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in roughly equal amounts were the major phospholipids. The membrane proteins were resolved into several major and minor protein bands of molecular weights ranging from 10,000 to 70,000 by acrylamide gel electrophoresis, and the protein pattern in the gel was different from those of the endoplasmic reticulum, mitochondrial, and glyoxysomal membranes.     

Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say)

Parasitization of newly ecdysed third, fourth, or fifth instar Manduca sexta larvae by the gregarious braconid wasp Cotesia congregata induces synthesis of new hemolymph proteins in the host. Analysis of hemolymph from parasitized and unparasitized control larvae using SDS gel electrophoresis showed that a major 33 kd band, plus several minor bands, were synthesized in parasitized but not control larvae; autoradiograms of proteins labeled in vivo for 1 hr with [³⁵S]methionine indicated that synthesis of the 33 kd polypeptide began a few hours following oviposition by the wasp. Synthesis of the 33 kd parasitism-specific polypeptide was induced in unparasitized larvae by the injection of ovarian calyx fluid from adult female wasps; this fluid is known to contain two morphologically distinct types of virus particles that are normally injected into the host along with eggs during parasitization. Exposure of calyx fluid to psoralen in the presence of long-wave u.v. light destroyed its capacity to induce synthesis of the 33 kd protein, suggesting that synthesis of this polypeptide may be mediated by viral nucleic acid.