Acid-stable protease inhibiting polypeptides formed from denatured horse plasma by proteolysis

1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. The Mr of the inhibitory polypeptides were 73,000 and 24,000. 3. The number, enzyme specificity and Mr of the inhibitory polypeptides differed from the values known for the human being. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose-trypsin and by gel filtration through Sephadex G-75. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, proteinase K, pronase, collagenase, papain and subtilisin. 6. The number and electrophoretic migration of the inhibitory polypeptides obtained with the different enzymes were variable. 7. The enzyme specificity was constant since all polypeptides inhibited only trypsin, chymotrypsin and kallikrein to a small extent. 8. None of the inhibitory polypeptides were immunologically related to native plasma proteins or plasma protease inhibitors.     

Polypeptide Synthesis in Simian Virus 5-Infected Cells

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.     

Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Temperature-sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. I. Identification by electrofocusing

The structural polypeptides of foot-and-mouth disease virus were analyzed by electrofocusing in a polyacrylamide gel containing 9 M urea. Three versions of the technique were used to accomodate the widely differing isoelectric points of the four polypeptides. VP2 was identified by comparing mature virus with procapsids. The selective actions of proteases on virions of two serotypes and on their 12S particles were examined. From this emerged a simple test for distinguishing the similarly sized polypeptides: VP1, VP2, and VP3. The effects of carbamylation and succinylation on the charge of the polypeptides were investigated. From the properties of polypeptides modified either chemically or by mutation, it was concluded that all amino acid substitutions that might be expected to cause a charge change would be detected except for neutral-to-histidine substitutions in the most basic polypeptide, VP1. In a sample of 73 temperature-sensitive mutants, 11 classes of variant polypeptides were distinguished on the basis of charge. Their molecular weights were unchanged. Alterations were found in all structural polypeptides except VP4. Mutations affecting VP2 caused similar shifts in the precursor, VP0.     

In vitro synthesis of peroxisomal membrane polypeptides

Peroxisomal membranes containing predominantly integral peroxisome membrane polypeptides were obtained from a highly purified peroxisomal fraction. Following sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and used to raise antibodies in rabbits. Cell-free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisomal membranes to reductive methylation [14C]-radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisome membrane polypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable presequences for these peroxisomal membrane polypeptides.     

Biochemical map of polypeptides specified by foot-and-mouth disease virus.

Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical polypeptides, were immunoprecipitated with the various antisera. To further establish identity, some proteins were compared by partial protease digestion. Evidence for a membrane association of the polypeptides coded for by the middle genome region is also presented. A biochemical map of the foot-and-mouth disease virus genome was established from the above information.     

The route of secretion of procollagen. The influence of alphaalpha''-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.     

Analysis of antigenic determinants of structural polypeptides of avian type C tumor viruses.

Radioimmunoassays were developed for the 19,000, 15,000, and 12,000 molecular weight polypeptides of avian myeloblastosis virus and for the 19,000 and 12,000 polypeptides of RAV-0, a subgroup E avian tumor virus. Each polypeptide was shown to possess both group- and type-specific antigenic determinants, in contrast to the 27,000 mol wt polypeptide, which contained only group-specific determinants. The corresponding low-molecular-weight polypeptides of subgroup A, B, and E viruses were shown to be immunologically indistinguishable. The findings that low-molecular-weight polypeptides of subgroup C and D viruses reacted very differently in immunoassays for the respective polypeptides of avian myeloblastosis virus or RAV-0 suggest that subgroups C and D may have evolved differently form subgroups A, B, and E.     

The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor-2 locus in barley (Hordeum vulgare L.)

Two-dimensional mapping (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the polypeptide components of “B” hordein fractions from eight barley varieties of widely different ancestry has been carried out. The relative positions of 47 different polypeptides were mapped, there being between 8 and 16 present in any one variety. The individual polypeptides differed in their distribution patterns; some were present in a number of varieties, while others were restricted to one or two. They also differed in their relative contributions to the total hordein fraction, both within and between varieties. The structural homology of the major polypeptides was compared by cleavage at methionine residues with cyanogen bromide and separation of the peptides on gradient gels. The polypeptides were classified into three groups which gave cleavage patterns with either two (class I), four (class II), or five (class III) low molecular weight bands. Class III polypeptides were found in all eight varieties, but in seven of the varieties class I or class II polypeptides were also present. With one exception, polypeptides migrating in the same position in different varieties gave identical or almost identical patterns. The three classes of polypeptides showed different distributions on the two-dimensional gels. Classes II and III polypeptides had a similar range of isoelectric points (pH 6.5–8.0), but all of the class II polypeptides were of slightly lower molecular weight. Class I polypeptides had a wider range of pI and molecular weight; the most alkaline and the lowest molecular weight polypeptides were in this group. The hordein fractions from a number of other barley varieties were compared with that of Julia. All had major polypeptides which migrated with ones present in Julia, but they differed in the relative amounts of these and in the absence of some polypeptides and the presence of others. B hordein is coded for by a single locus which has been suggested to be a complex multigenic family derived by duplication and divergence of a single gene. The data reported here provide support for this hypothesis and suggest that both mutations in the duplicated genes and recombination within the locus may have contributed to the polymorphism of the polypeptides.     

Identification of bovine sperm specific polypeptides reactive with antisperm antibodies.

The present study was taken to characterize molecular weights of sperm specific polypeptides antigenic to rabbits and calf with the aim to assess their immunoreactivity with IgG antibodies in sera from immuno-infertile cows. Seropositivity for antisperm IgG antibodies in 75 repeat breeder and 15 pregnant control cattle was tested by cellular ELISA using washed spermatozoa antigen from 4 bulls. Molecular weights of bovine sperm polypeptides antigenic to rabbit and calf were determined by 10% SDS-PAGE and Western blotting. Molecular weights of sperm peptides reactive with sera from immuno-infertile cows were also determined. Seropositivity of antisperm IgG antibodies for bull I, II, III and IV was 23.6, 14.6, 26.6 and 20%, respectively. A total of 16 polypeptides were discernible on gel. Out of these, 7 polypeptides were immunoreactive with sera from hyperimmunized rabbits as compared to 3 poly-peptides which reacted with sera from hyper-immunized calf. Only two polypeptides were reactive with sera from immuno-infertile cows. Variable number of sperm polypeptides and their immunoreactivity have been reported in different species. Antigenicity of different polypeptides in sperm needs further investigations.     

Characterization of seminal plasma proteins and sperm proteins in ejaculates from normospermic bulls and bulls with thermally-induced testicular degeneration

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.     

Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.     

Purification of membrane polypeptides of rat liver peroxisomes

Peroxisomes were obtained by sucrose density gradient centrifugation from the livers of di(2-ethylhexyl)phthalate-fed rats, and the membranes were prepared by carbonate extraction (Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., & Lazarow, P.B. (1982) J. Cell Biol. 93, 103-110). The integrated membrane polypeptides were solubilized with sodium dodecyl sulfate, and purified by repeated polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of 70 and 68 kDa polypeptides was not attempted in the present study because of their close migration in polyacrylamide gel electrophoresis. Other polypeptides with apparent molecular masses of 41, 27, 26, and 22 kDa were purified to near homogeneity. Antibodies were raised against these purified preparations. The 68 kDa polypeptide is suggested to be produced by the proteolytic modification of 70 kDa polypeptide, since the former increased concomitantly with decrease of the latter when the liver homogenate was incubated, and this change was prevented in the presence of leupeptin during the incubation. The 41 kDa polypeptide was a minor component. The 70 and 68 kDa polypeptides and 41 kDa polypeptide and their antibodies were cross-reactive, but the relation of these polypeptides was not clear. The 27 and 26 kDa polypeptides seemed to be another species of membrane polypeptides, although the relationship of these two polypeptides remains to be clarified. The 22 kDa polypeptide is not related to other membrane polypeptides. The results of immunoblot analysis of subcellular fractions of the liver and an electron microscopic immunocytochemical study to locate the antigenic sites with protein A-gold complex suggest that all of these polypeptides are localized on peroxisomal membranes. On proliferation of rat liver peroxisomes by administration of di(2-ethylhexyl)phthalate, a peroxisome proliferator, all of these polypeptides were markedly increased.     

Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa.

In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical.     

Biosynthesis of membrane polypeptides of rat liver peroxisomes

The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.     

Analysis of the polypeptide composition of grana and stroma thylakoids by two-dimensional gel electrophoresis. Distribution of photosystem II between grana and stroma lamellae

The polypeptide composition of whole thylakoids and membrane subfragments was studied by using a modified two-dimensional gel electrophoresis technique of O'Farrell [J. Biol. Chem. 250, 4007-4021 (1975)]. The modifications were lithium dodecyl sulphate solubilization instead instead of SDS, reverse isofocusing and sensitive silver staining procedure. This high-resolution technique allowed us to separate and identify about 170 polypeptides of thylakoid membranes. After separating grana and stroma thylakoids it was found that both types of lamellae contained nearly equal amounts of polypeptides, but about 70 polypeptides were different in the two preparations. In grana thylakoids, 54 polypeptides out of 95 were found to be mainly present in grana and 31 of them were only present in grana preparations. In stroma membranes, 43 polypeptides out of 99 were mainly present in stroma lamellae and 38 of these polypeptides were exclusively present in stroma lamellae. In a functional photosystem II preparation, 61 individual polypeptides could be distinguished. Most of these polypeptides were present in both grana and stroma lamellae, but 22 of them were more pronounced in grana than in stroma lamellae. 9 polypeptides of photosystem II were distinctly different in grana and stroma lamellae, and these differences may connect closely with the functional differences of photosystem II in the two types of thylakoids.     

Uptake and incorporation of the products of proteolysis by the rumen bacteriumBacteroides ruminicola R8/4

The polypeptides of the proteolytic rumen bacteriumBacteroides ruminicola R8/4 grown in the presence of either leaf Fraction 1 protein, bovine serum albumin, or Bactocasitone as sole nitrogen source were separated by SDS-polyacrylamide gel electrophoresis. Over 40 polypeptides were resolved; the pattern for organisms grown on Fraction 1 protein was similar but not identical to that of the serum albumin and Bactocasitone-grown bacteria. All the bacterial polypeptides were distinguishable from the polypeptides of Fraction 1 protein (and serum albumin). The stained pattern was the same for organisms sampled at intervals during the growth of a batch culture. After incubation of the growing organisms with [¹⁴C]-Fraction 1 protein, all the bacterial polypeptides were labeled. Bacteria grown in the presence of nonlabeled Fraction 1 protein and a mixture of [^14C]-labeled amino acids incorporated label into all the polypeptides; the bacteria did not grow in the absence of intact protein, and then virtually no label was incorporated from the amino acid mixture.