High-voltage Paper Electrophoresis Research Articles

Evidence for the vitamin K-dependent gamma-carboxylation of the first glutamic acid residue in peptide substrates containing a diglutamyl sequence.

The peptide substrate commonly used in vitamin K-dependent carboxylation, Phe-Leu-Glu-Glu-Val, has been shown, by the use of high-voltage paper electrophoresis, to be degraded from the N-terminus by a microsomal leucine amino-peptidase. The replacement of phenylalanine with a N-t-butoxycarbonyl group resulted in a tetrapeptide substrate with a blocked N-terminus resistant to enzymic degradation. Vitamin K-dependent carboxylation of this non-degradable substrate gave a unique carboxylated product, which was separated from microsomal protein and unchanged substrate by using DEAE-Sephadex A25 as a final step. The carboxylated product was subsequently decarboxylated in 2HCl and analysed by using g.l.c. coupled to a mass spectrometer. This showed that only the first glutamic acid residue in the peptide substrate was carboxylated.

Intrarenal angiotensin I conversion at normal and reduced renal blood flow in the dog.

Intrarenal conversion of angiotensin I (ANG I) to angiotensin II (ANG II) under conditions of normal and reduced renal blood flow (RBF) elicited by constriction of the renal artery was examined in pentobarbital-anesthetized dogs. In eight animals, tracer doses of 125I-ANG I (5-12 pmol) were injected into the renal artery and 125I-ANG I, 125I-ANG II, and 125I-labeled metabolites were measured in renal venous effluent by high-voltage paper electrophoresis. The mean conversion of ANG I to ANG II during a single passage through the kidney was 21.8 +/- 2.1% at control RBF. When RBF was decreased by 25 and 53%, percent ANG I conversion was not altered significantly. In six dogs percent conversion of 125I-[Sar1, Ile5]ANG I, an ANG I analogue refractory to hydrolysis by aminopeptidases, was 18.1 +/- 1.7% at control RBF and did not change significantly when the RBF was reduced by 55%. Although there were severalfold increases in renal renin secretion rate and net ANG I generation rate during reduced RBF, net renal ANG II formation rate did not change significantly. These data indicate that there is substantial conversion of ANG I in a single passage through the dog kidney and that intrarenal ANG I conversion is independent of RBF even under conditions in which renin secretion rate and ANG I generation rate are increased severalfold.

Beta-Crystallin: endogenous substrate of lens transglutaminase. Characterization of the acyl-donor site in the beta Bp chain.

Incubation of calf lens cortex homogenate with [14C]putrescine or dansylcadaverine, followed by two-dimensional gel electrophoresis and fluorography, enabled the identification of three different beta-crystallin chains as the endogenous substrates of Ca2+-dependent lens transglutaminase (R-glutaminyl-peptide:amine-gamma-glutamylyltransferase, EC 2.3.2.13). One of these is beta Bp, the predominant subunit of beta-crystallin, of which the amino acid sequence is known. The site of amine-labeling in beta Bp could be located, by limited proteolysis, in the N-terminal domain of this chain. Tryptic digestion of the N-terminal domain and subdigestion with elastase of the N-terminal tryptic peptide identified glutamine-7 as the single residue to which the amines are bound. This is the first example of an endogenous substrate of intracellular transglutaminase in which the site of the acyl-donor glutamine residue has been established. Tryptic digestion of the putrescine-labeled beta-crystallin aggregate, followed by high-voltage paper electrophoresis, provided a preliminary characterization of the labeled peptides originating from the other two labeled beta subunits.

Multi-site phosphorylation in ox-kidney branched-chain 2-oxoacid dehydrogenase complex

Tryptic [ 32P]phosphopeptides were prepared from [ 32P]phosphorylated ox-kidney branched-chain complex and analysed by high-voltage paper electrophoresis at pH 1.9. In the maximally phosphorylated complex 3 tryptic [ 32P]phosphopeptides were identified (TA, TB, TC). R F-values relative to N 6-dinitrophenyllysine were (mean ± SEM for 25 obs.): TA, 1.53 ± 0.03; TB, 1.07 ± 0.02; TC, 0.65 ± 0.01. Relative rates of phosphorylation were TA > TB > TC. Phosphorylation of TA reached a maximum when about 66% of the complex was inactivated. Phosphorylation of TB and TC was associated mainly with 66–95% inactivation of the complex.

Evidence for a β-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO(4) at pH 6.5 and then treated with sodium [(3)H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110 degrees C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [(3)H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the beta-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.

Preparation of peptides containing any desired amino acid: methionyl peptides of bovine rhodopsin.

A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.

End group analysis of the cytosolic and mitochondrial fumarases from rat liver.

Some molecular properties of the cytosolic and mitochondrial fumarases were compared. The carboxyl(C)-terminal amino acid of both the cytosolic and mitochondrial fumarases of rat liver cell was identified as leucine by using carboxypeptidase (CPase) A. As the amino(N)-terminal amino acid of both the cytosolic and mitochondrial fumarases could not be identified by the dansyl chloride method or by the cyanate method, the N-termini of these two fumarases seems to be masked. Both fumarases, after S-carboxymethylation, were completely digested with pronase E and CPase A and B, and the amino acids with blocked amino group were analyzed by high voltage paper electrophoresis and amino acid analysis after acid hydrolysis of these amino acid derivatives. The N-termini of the mitochondrial and cytosolic fumarases were identified as pyroglutamic acid and N-acetylalanine, respectively. To compare the primary structures of the two fumarases in detail, each fumarase was digested with an arginine-specific protease or cleaved with cyanogen bromide. The electrophoretic profiles of the digests of these fumarases were indistinguishable from each other.

Disulfide bridges in an alpha-amylase inhibitor from wheat kernel.

An alpha-amylase inhibitor (0.53-inhibitor) was digested with pepsin and the cystine-containing peptides were isolated by SP-Sephadex C-25 column chromatography and high voltage paper electrophoresis. Amino acid compositions and partial sequences of these peptides and amino acid compositions of the peptides separated after performic acid oxidation revealed the presence of 4 disulfide bonds in the inhibitor. They were formed between residues 6 and 115, 20 and either 41 or 42, 99 and either 41 or 42, and 28 and 83. The disulfide bond partners of residues 20 and 99 were not conclusively determined because of the difficulty of cleavage of the Cys-Cys bond at residues 41 and 42. Among 9 cysteines in this inhibitor Cys-54 must be in a free form, since no evidence was obtained for its contribution to disulfide bond formation.

A rapid and sensitive paper electrophoresis assay for the detection of microbial siderophores elicited in solid-plating culture

A rapid and sensitive assay for the detection of microbial siderophores (iron-binding compounds) is described. Nine representative fungal and bacterial cultures including Ustilago sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae, Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by sequential transfers on iron-deficient solid-plating media. In response to Fe-stress conditions, the microorganisms excreted siderophore compounds into the extracellular solid culture medium. The solid agar matrix effectively concentrated and restricted the migration of the siderophore compounds to the region immediately adjacent to colonial growth. Agar-block samples from this region were removed and placed at the origin of an electrophoresis paper strip. The resultant absorbed material from the agar-block sample was subjected to high-voltage paper electrophoresis which separated the siderophore compounds by size and molecular net charge. Phenolic acid (“catechol”)-type siderophores were detected by fluorescence under uv light. Hydroxamic acid-type siderophores were visualized by spraying the electrophoretogram with ferric iron solution.

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230 000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.

Isolation of a 3-deoxy-D-mannooctulosonic acid disaccharide from Salmonella minnesota rough-form lipopolysaccharides.

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70 degrees C/3 h. After dialysis of the hydrolysates about one third of the total 3-deoxy-D-mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate. By high-voltage paper electrophoresis, a compound with the mobility of 1.2 relative to dOclA could be isolated from the dialysate. It was identified as a dOclA disaccharide by hydrolysis without or after reduction with sodium borohydride and by analysis with the thiobarbituric acid assay under different conditions. The ketosidic linkage in the disaccharide is assumed to be 2.4 or 2.5.

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls. Fingerprint analysis revealed that the same sequences of mengovirus RNA were protected against nuclease attack by the 80S and the 40S initiation complexes formed in vitro in lysates of control or interferon-treated L-cells. Mengovirus RNA-coded proteins were labeled at their N-terminal end with formyl-[35S]methionine and digested to completion with trypsin. The resulting fragments were separated by high-voltage paper electrophoresis. Two different formyl-[35S]methionine-labeled N termini were resolved. Further analyses supported the notion that the two radioactive peaks originated in the initiation of translation at two different sites. This pattern did not change when mengovirus RNA was translated in lysates of interferon-treated cells.

Relationship between H1 histone phosphorylation and genome replication in a mouse-Chinese hamster somatic cell hybrid

The relationship between stable genome replication and the presence of species-specific H1 and H2b histones was studied in a mouse-Chinese hamster somatic cell hybrid (LMC-1). LMC-1 contained the complete chromosome complements of the mouse ( LM TK − , 46 chromosomes) and Chinese hamster (CHW-1102, 22 chromosomes) parents. No chromosome loss occurred in over a year in continuous culture, and both mouse and Chinese hamster enzymes were expressed. Chromatographic resolution of H1 histone subtypes showed that only mouse H1 genes were expressed. Resolution of chromatographically purified H2b histone variants using Triton X-100-containing gels also demonstrated that LMC-1 cells made mouse, not Chinese hamster, H2b histones. Species-specific tryptic phosphopeptides were resolved from purified H1 histones using high voltage paper electrophoresis. These studies demonstrated that characteristic mouse, but not Chinese hamster, phosphopeptides were formed in H1 histones in actively-growing LMC-1 cells. We conclude that the Chinese hamster genome may replicate in a stable manner in the presence of mouse H1 (and H2b) histones, and that there is no obligatory relationship between species-specific phosphorylation of H1 histones and stable genome replication.

The conversion of met-enkephalin-Arg 6, Phe 7 to met-enkephalin in rabbit ear artery

We studied the opioid agonist activities of met-enkephalin-Arg 6, Phe 7 heptapeptide (ME-A,P) and its D-Ala 2-analogue in several isolated organs including rabbit ear artery (ART), guinea-pig ileum (GPI) mouse-, rat and rabbit vas deferens preparation. Both heptapeptides followed the in vitro activity pattern of met-enkephalin (ME). There was a significant parallelism in the sensitivity of ART and mouse vas deferens to opioid peptides. In ART, ME-A,P must be converted by a captopril sensitive enzyme to exert opioid agonist action. Incubation of heptapeptide with membranes prepared from rabbit ear artery revealed the captopril-sensitive enzymic conversion of ME-AP to ME as detected by high voltage paper electrophoresis.

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor-bound neurotoxin

Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50–150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, when the toxin binds to the receptor. The corresponding values for the other amino acids are: Ac-Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residues Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.

Determination of β-galactosidase activity in the intestinal tract of mice by ion-exchange high-performance liquid chromatography using ϵ-N-1-(1-deoxylactulosyl)- L-lysine as substrate

ϵ-N-1-(1-Deoxylactulosyl)- L-lysine was synthesized and used as a substrate to assay β-galactosidase activity. ϵ-N-1-(1-Deoxylactulosyl)- L-lysine and its degradation product ϵ-N-1-(1-deoxyfructosyl)- L-lysine were detected by high-voltage paper electrophoresis and ion-exchange high-performance liquid chromatography. The β-galactosidase activity in different parts of the intestinal tract of germ-free and control mice was determined and compared with a β-galactosidase activity which degrades lactose at pH 8.5 and 5.0 and which corresponded with bacterial and host enzymatic activities, respectively.

Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system

In vitro biosynthesis of neurosecretory peptides specific to the X-organ sinus gland (XOSG) of the land crabCardisoma carnifex was studied. The neurosecretory somata comprising the X-organ were selectively exposed to saline containing3H-leucine, glucose and antibiotics for 5 h (pulse) followed by 19–24 h of superfusion with nutrient media (chase). Analysis of extracts of somata and terminal regions by high voltage paper electrophoresis demonstrated the uptake and incorporation of3H-leucine into protein by X-organ somata and its axonal transport to terminal regions. Tritiated-leucine incorporation was reduced 90% by 50 μg/ml puromycin, indicating ribosome-mediated synthesis. Axonal transport was blocked by ligation of the axons; this did not affect the rate of3H-leucine incorporation by the somata. Radiolabelled peptide hormones were identified by comigration of radiolabel with RPLC and/or SDS-PAGE determined regions of bioactivity and by comparison of RPLC elution patterns with those of sinus gland peptides purified and analyzed for amino acid composition by Newcomb (1983a). Erythrophore concentrating hormone (ECH) and at least two 6,000 Dalton peptides having crustacean hyperglycemic hormone activity (CHH) were identified among peptides biosynthetically radiolabelled. Comparison of XOSG systems receiving 5 h of incubation with those receiving a pulse-chase (5 h∶19 h) regime demonstrates the existence of polypeptide precursors to the XOSG peptide hormones.

Elucidation of the disulfide bond positions of the β-subunit of human follicle-stimulating hormone

From a proteolytic digest of human follicle-stimulating hormone (FSH), three of the five disulfide bonds in the α-subunit of human FSH, namely, those between half-cystine residues at positions 7–10, 28–87 and 82–84, and three of the six disulfide bonds in the β-subunit of human FSH, namely, those between half-cystine residues at positions 3–28, 17–51 and 32–104, have been previously identified. The remaining fractions of the proteolytic digest containing the rest of the cystine peptides of human FSH were pooled, and fractionated by gel filtration on Sephadex G-50 (superfine) and by high-pressure liquid chromatography. One high molecular weight fragment containing half-cystine residues was recovered. The fragment was resistant to a sequential digestion with specific proteolytic enzymes, such as thermolysin and l-(1-tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin. This fragment, on the basis of amino acid analysis, N- and C-terminal and limited amino acid sequence analysis, was identified as a single peptide containing the remaining six half-cystine residues or three disulfide bonds of the human FSH β-subunit. Hence, the fragment was digested with a mixture of proteases such as pronase, papain, subtilisin, chymotrypsin, thermolysin and TPCK-treated trypsin, in order to achieve a random cleavage of the peptide. From this enzyme digest, two cystine-containing peptides were isolated by gel filtration on Sephadex G-50, and high-voltage paper electrophoresis at pH 2 and pH 3.5. The peptides were identified by amino acid, N- and C-terminal and sequence analyses as containing disulfide bonds between βCys-66-βCys-94 and βCys-20-βCys-82. The single remaining bond was assigned between βCys-84-βCys-87 by elimination, thus completing the identification of all six disulfide bonds of the β-subunit of human FSH.

Comparison of methods for the determination of β-aspartylglycine in fecal supernatants of leukemic patients treated with antimicrobial agents

β-Aspartylglycine is an indicator of the absence of bacterial enzymatic activity in the intestinal tract. This study describes and compares four methods — (1) dansylation with thin-layer chromatography, (2) ion-exchange chromatography, (3) thin-layer electrophoresis, (4) high-voltage paper electrophoresis — to determine the concentration of β-aspartylglycine in fecal supernatants of leukemic patients treated with antimicrobial agents.

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides.

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.