Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Effects of Temperature and Nutrients on Proteinase Production by Pseudomonas fluorescens and Ps. aeruginosa in Broth and Milk

Temperature and the composition of the medium influenced the production of proteinase by Pseudomonas fluorescens and Pseudomonas aeruginosa isolated from raw milk. Many isolates of Ps. fluorescens digested litmus milk at 10° but not at 5° or 2°. With Ps. fluorescens proteinase production per unit of growth in a Peptone–Yeast Extract broth declined progressively as the incubation temperature was reduced from 20° to 5°. At 30° there was heavy growth in the same medium but only slight proteinase production whereas enzyme production by Ps. aeruginosa was maximal at this temperature. Proteinase production by both species in semi-defined media was essentially a function of the organic nitrogen content of the medium; there was evidence of catabolite repression by glucose and, to a lesser extent, lactate. In milks seeded with these pseudomonads, the extent of proteolysis was either increased markedly or slightly decreased when glucose was included.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Comparative studies of two types of acid proteases from rat gastric mucosa and spleen

Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; these two types of acid proteases are quite different proteins existing in the individual tissues.     

Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections

The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist.     

Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR

To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.     

Carbohydrate utilization affects Lactobacillus delbrueckii subsp. lactis 313 cell-enveloped-associated proteinase production

The effect of different sugars (glucose, glycerol, maltose, galactose and lactose) on cell-membrane-associated proteinase production by Lactobacillus delbrueckii subsp. lactis 313 (LDL 313) was investigated. The experimental results showed that aside glycerol and galactose, all the other sugars supported high growth levels of LDL 313, with glucose displaying the maximum biomass concentration of 0.85 mg/mL dry cell weight for cells harvested at the mid-exponential phase of ??12 h after inoculation. The specific proteinase yield, a measure of the rate of proteinase production relative to cell wall biosynthesis, was used to evaluate the preferential degree of proteinase metabolism as induced by the consumption of different sugar substrates by LDL 313. It was found that maltose displayed the highest specific proteinase yield of 12.59 U/mg sugar consumed. Further, molecular differences were observed in the SDS electrophoretic profile of cell surface proteins generated for the different carbon substrates. This is a preliminary study which supports the inference that different sugars stimulate the production of different cell-surface proteins with a significant effect on cell proteinase activity.     

Influence of sodium dodecyl sulphate quality on the electrophoretic mobility of the outer membrane proteins of mucoid and non-mucoid Pseudomonas aeruginosa

The electrophoretic mobilities of proteins F and H1 from the outer membrane of Pseudomonas aeruginosa (mucoid and non-mucoid) in polyacrylamide gel electrophoresis were affected by the quality of sodium dodecyl sulphate (SDS) used. In particular, the sodium tetradecyl sulphate impurity present in crude SDS influenced the mobilities of F and H1. These observations explain conflicting reports on changes in outer membrane proteins with strain and growth conditions. Synthesis of H1 was induced by growth in magnesium depleted medium but repressed when calcium or manganese were added to magnesium depleted medium.     

Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms.

Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl substrate were attacked most rapidly and their hydrolyses were stimulated by Ca2+. The 2-naphthylamide derivatives of neutral and basic amino acids were also hydrolysed by aminopeptidase A, but at rates about two orders of magnitude lower, and Ca2+ was inhibitory. The possibility that these atypical substrates were hydrolysed by traces of aminopeptidase M (EC 3.4.11.2) contaminating the preparation could be excluded on several grounds. Aminopeptidase A was sensitive to inhibition by chelating agents and the inactive enzyme could be reactivated by Ca2+ or Mn2+. Atomic absorption spectrophotometry revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar protein.     

Interactions of the Integrase Protein of the Conjugative Transposon Tn916 with Its Specific DNA Binding Sites

The binding of two chimeric proteins, consisting of the N-terminal or C-terminal DNA binding domain of Tn916 Int fused to maltose binding protein, to specific oligonucleotide substrates was analyzed by gel mobility shift assay. The chimeric protein with the N-terminal domain formed two complexes of different electrophoretic mobilities. The faster-moving complex, whose formation displayed no cooperativity, contained two protein monomers bound to a single DNA molecule. The slower-moving complex, whose formation involved cooperative binding (Hill coefficient > 1.0), contained four protein monomers bound to a single DNA molecule. Methylation interference experiments coupled with the analysis of protein binding to mutant oligonucleotide substrates showed that formation of the faster-moving complex containing two protein monomers required the presence of two 11-bp direct repeats (called DR2) in direct orientation. Formation of the slower-moving complex required only a single DR2 repeat. Binding of the N-terminal domains in vivo could serve to position two Int monomers on the DNA near each end of the transposon and assist in bringing together the ends of the transposon so that excision can occur. The chimeric protein with the C-terminal domain of Int also formed two complexes of different electrophoretic mobilities. The major, slower-moving complex, whose formation involved cooperative binding, contained two protein molecules bound to one DNA molecule. This finding suggested that while the C-terminal domain of Int can bind DNA as a monomer, a cooperative interaction between two monomers of the C-terminal domain may help to bring the ends of the transposon together during excision.     

Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions

Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.     

Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.     

Influence of sodium dodecyl sulphate quality on the electrophoretic mobility of the outer membrane proteins of mucoid and non-mucoid Psedomonas aeruginosa

The electrophoretic mobilities of proteins F and H1 from the outer membrane of Pseudomonas aeruginosa (mucoid and non-mucoid) in polyacrylamide gel electrophoresis were affected by the quality of sodium dodecyl sulphate (SDS) used. In particular, the sodium tetradecyl sulphate impurity present in crude SDS influenced the mobilities of F and H1. These observations explain conflicting reports on changes in outer membrane proteins with strain and growth conditions. Synthesis of H1 was induced by growth in magnesium depleted medium but repressed when calcium or manganese were added to magnesium depleted medium.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions

Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme-linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages.     

Microspectrophotometric and enzymatic analyses of fish plasma proteins electrophoretically separated in thin polyacrylamide gels

A system for horizontal, discontinuous electrophoresis in thin sheets of polyacrylamide gel was developed to permit rapid and direct comparison of multiple samples of fish plasmas for population studies. The resulting electropherograms are suitable either for screening for enzyme polymorphisms or for densitometric scanning after gels are stained for total proteins. Each gel sheet (110×90×0.8 mm) can be used for simultaneous separation of 10–15 different samples. Since total voltage and the running time required for successful resolution of fine protein bands are greatly reduced in this system, minimal heating occurs within the gel matrix during electrophoresis. Up to 120 different blood samples (8 gel sheets) can be processed in about 8 h. Gel sheets are routinely stained overnight in Coomassie Brilliant Blue (0.1%, v/v), rinsed several times and stored overnight in 7% acetic acid to complete destaining. Particular gel sectors are then transferred to distilled water and mounted on glass microslides for photography or for densitometric evaluation with a Leitz microspectrophotometer. These procedures have been used to identify characteristic differences in the electrophoretic mobilities of plasma enzymes and albumin fractions from several populations of poeciliid fishes. Observed differences in albumin mobilities (albumin phenotypes) were verified by mixing isoaliquots of test plasmas with plasma samples containing albumins of known mobility. Resultant patterns for albumin bands for such mixed plasmas were indistinguishable from those obtained with plasma samples from the F₁ hybrid progeny of parents possessing albumins of characteristically different electrophoretic mobilities. Procedural details for gel casting, electrophoresis and sample evaluation are described.     

Comparative zone electrophoresis of catalase of Staphylococcus species isolated from mammalian skin.

Vertical polyacrylamide slab gel electrophoresis was conducted for the catalase enzymes of representative strains of 18 proposed species and subspecies of the genus Staphylococcus. The catalase bands which resulted were predominantly monomorphic within each of the species and differences in catalase mobilities were observed between many of the species. The electrophoretic mobilities of the catalases were supportive to the scheme of classification used. Many strains of certain species demonstrated multiple catalase bands which are suggestive of multimolecular forms of the enzyme. Horizontal starch gel electrophoresis of representative strains of S. capitis produced catalase bands with relative mobilities that were different from those obtained with polyacrylamide electrophoresis, presumably due to a difference in molecular sieving between the gels.     

Effect of ribonuclease and neuraminidase on the electrophoretic mobility of tissue culture cells in parasynchronous growth

Cultured mammalian cells (RPMI no. 41) in parasynchronous growth were treated, at different stages of the mitotic cycle, with neuraminidase and ribonuclease, separately and sequentially, and their electrophoretic mobilities determined. Changes in the electrophoretic mobility of these cells are probably mainly due to variations in the density of negatively charged groups susceptible to neuraminidase, although variations in groups susceptible to ribonuclease may occur. It is suggested that the observed variations in electrophoretic mobility of different cells may be due to differences in the relative lengths of different life-cycle phases. Where G₂ phase is relatively long or G₁ relatively short the cell populations will hve higher mean electrophoretic mobilities.