Indentification of two somatomedin A active polypeptides and in vivo effects of a somatomedin A concentrate

The first chemical characterization of two polypeptides from human serum which stimulate the $\text{in vitro}$ incorporation of ³⁵S sulfate into chick cartilage is described. These two polypeptides, designated Somatomedin A₁ and A₂ have a molecular weight of approximately 7000. Although each peptide contains 1 cysteine residue and has asparagine as amino terminal residue, there are apparent differences in the amino acid composition. Administration of a Somatomedin A concentrate to hypophysectomized rats gave an increase in tibial width similar to that obtained with 20 μg human growth hormone.     

In vivo incorporation of different amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria

The $\text{in vivo}$ incorporation of each of the twenty common amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria has been investigated. Under labeling conditions which allow translation only on mitochondrial ribosomes, incorporation of all the amino acids, except aspartic acid, cysteine, glutamic acid and glycine, has been detected. These exceptions are probably due to problems related to amino acid pool size and/or equilibration.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Anti-glycosyl antibodies: Preparation and characterization of rabbit anti-galactose and anti-lactose antibodies

Anti-galactose and anti-lactose antibodies have been isolated from the antisera of rabbits immunized with non-viable cells of $\text{Streptococcus}$$\text{faecalis}$, strain N containing an antigenic diheteroglycan of glucose and galactose in the cell wall. The anti-galactose antibodies are specific for the galactosyl moiety while the anti-lactose antibodies are specific for the lactosyl moiety of the diheteroglycan. Hapten inhibitions with galactose and lactose, the sedimentation constant, the immunoglobulin type, the carbohydrate content, the electrophoretic mobility and the amino acid composition have been determined for the two new types of anti-glycosyl antibodies.     

Amino terminal amino acid sequence of the major polypeptide subunit of Torpedo californica acetylcholine receptor

The amino terminal sequence of the 40,000 dalton polypeptide subunit of $\text{Torpedo}\text{californica}$ acetylcholine receptor has been determined for twenty-five cycles using automatic microsequencing procedures. The results demonstrate a unique polypeptide sequence for this receptor subunit and quantitation of amino acid recoveries shows that no significant amounts of polypeptides with blocked amino terminii are present.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Bacteriophage-induced lytic enzyme which hydrolyzes L-alanine-D-glutamic acid peptide bond in peptidoglycan

The mode of action of bacteriophage-induced lytic enzyme “LE95” was investigated. The LE95 hydrolyzed peptide portion in peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The exposed amino terminal amino acid was identified as glutamic acid by analysis of terminal amino acid by dinitrophenylation. This result suggested the LE95 hydrolyzed the peptide bond between L-alanine and D-glutamic acid in the peptidoglycan of $\text{Ps. aeruginosa}$ and $\text{E. coli}$. The enzyme did not hydrolyze various peptides prepared from bacterial cell wall. This experimental result suggested that the glycan chain of peptidoglycan would be essential for the enzymic activity.     

In vitro synthesis of nerve growth factor related glioma C6 cell polypeptides.

Rat glioma C₆ cell polyribosomal preparations were tested in a heterologous $\text{in vitro}$ system for their ability to direct the synthesis of nerve growth factor related polypeptides. Two major polypeptides of MW ～ 21,000 and ～ 43,000 respectively were found, both of which were immunoprecipitable with specific anti-mouse 2.5S nerve growth factor serum. After incubation of $\text{in vitro}$ synthesized proteins with submaxillary gland extract the bulk of these protein species was converted into immunoprecipitable material of MW ～ 13,000, which comigrated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis with mouse 2.5S nerve growth factor.     

Purification of penicillin-insensitive DD-endopeptidase, a new cell wall peptidoglycan-hydrolyzing enzyme in Escherichia coli, and its inhibition by deoxyribonucleic acids.

Activity of a penicillin-insensitive $\text{DD}$-endopeptidase that splits the $\text{D}$-alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of $\text{Escherichia coli}$. The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments.     

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.     

Interaction of phenol with the polysaccharide of bacteria cell wall

Phenol forms an insoluble complex with the polysaccharide in the cell wall of $\text{M.}$$\text{lysodeicticus}$, and renders it resistant to the action of lysozyme. The formation of the stable complex in the wall accounts for the bactericidal action of phenol. The application of lysozyme in this study affords a unique means of measuring the rate and degree of reaction between phenol and the polysaccharide.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Studies on the purification and properties of teleost prolactin

A protein fraction containing prolactin activity from the pituitary tissue of a teleost fish, $\text{Tilapia mossambica}$, has been purified by a combination of ion-exchange and exclusion chromatographic procedures. The purified $\text{Tilapia}$ prolactin was characterized by disc gel electrophoresis, amino-terminal group identification, and amino acid analysis. Its amino acid composition was found to be similar to ovine prolactin. The purified fish prolactin was found to be 40–50 times more potent than ovine prolactin in the $\text{Tilapia}$ sodium-retaining bioassay. However, it was found to be devoid of $\text{Gillichthys}$ yellow pigment-dispersing activity which was previously thought to be a property of teleost prolactin.     

Changes in order of migration of polypeptides in complex III and cytochrome C oxidase under different conditions of SDS polyacrylamide gel electrophoresis.

The order of migration of polypeptides in both cytochrome $\text{c}$ oxidase and ubiquinone cytochrome $\text{c}$ reductase has been found to differ depending on the gel conditions used. Thus the nomenclatures or numbering systems being used for the subunits of these membrane complexes by workers using Weber-Osborn gels is not the same as that being used in laboratories which use the Swank-Munkres or Fairbanks gel procedure.     

Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations

We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, $\text{N-}\text{ethylmaleimide}$ treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in $\text{N-}\text{ethylmaleimide-treated}$ cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis.The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292–313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.     

Degradation of surface-labeled hepatoma membrane polypeptides: Effect of inhibitors

When their membrane proteins were labeled with ¹²⁵I by lactoperoxidase, dividing hepatomacells lost radio activity to the medium in a biphasic manner ($\text{T}\text{1}\text{2}\text{= 16–26}\text{h}\text{, > 40}\text{h}$). Lysosomotropic weak bases, chloroquine, and NH₄Cl inhibited the rapid phase by 59%. More than 50% of the radio activity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60–71% inhibited by lysosomotropic agents chloro quine and NH₄Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH₄Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-l-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by21–24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 °C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lyososomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to, agents which disrupt the cytoskeleton.     

Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds

Lipoteichoic acid (LTA) from several Gram-positive microorganisms, Forssman antigen (Fag) from $\text{Diplococcus pneumoniae}$ R36A, and an acidic lipopolysaccharide (ALP) from $\text{Micrococcus luteus}$ were examined for effects on four wall lysis systems. The LTAs inhibited the N-acetylmuramidases of $\text{Streptococcus faecalis}$ and $\text{Lactobacillus acidophilus}$, and the amidase of $\text{Bacillus subtilis}$ 168. Deacylated LTA failed to inhibit. LTAs failed to inhibit the amidase of pneumococcus. Fag inhibited the pneumococcal amidase, but had no effect on the other three systems.     

Biosynthesis of glycerol teichoic acid in Bacillus cereus: formation of linkage unit disaccharide on a lipid intermediate

A tunicamycin-like antibiotic 24010 at a concentration of 1 μg/ml selectively inhibited the $\text{in vivo}$ synthesis of glycerol teichoic acid of cell walls in $\text{Bacillus cereus}$ AHU 1030. Incubation of membranes of this strain with $\text{N}$-acetylglucosaminyl pyrophosphorylundecaprenol and UDP-$\text{N}$-acetylmannosamine led to formation of a glycolipid having a saccharide moiety identical with the cell wall teichoic acid linkage unit, $\text{N}$-acetylmannosaminylβ(1→4)-$\text{N}$-acetylglucosamine. The membranes also catalyzed transfer of glycerol phosphate units from CDP-glycerol to this disaccharide-linked lipid. Thus the biosynthesis of the cell wall glycerol teichoic acid in this strain seems to involve the disaccharide-linked lipid as an intermediate.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.