Genetic polymorphism of the vitamin D binding protein (Gc) in the musk shrew (Suncus murinus)

In the musk shrew ( Suncus murinus ), the electrophoretic bands in the post-albumin region were identified as vitamin D binding protein (Gc) by the [³HI vitamin D₃ binding method. Three Gc phenotypes were distinguished from each other: a single faster band (Gc-A), a single slower band (Gc-B) and the double bands (Gc-AB). Results of mating experiments indicated that the Gc-A and Gc-B are controlled by two codominant alleles, Cc ^a and Gc ^b at an autosomal locus ( Cc ), respectively. It was noticed that, in the Gc-AB phenotypes, the Gc-B band was constantly more intense than the Gc-A band in the protein staining. The same tendency was also observed btween the homozygous Gc-A and Gc-B bands, and further, radioactivity of the Gc-B bound with [³H] vitamin D₃ was about twofold higher than that of the Gc-A. These results suggest that the Gc^b yields its protein product twofold more than the Gc ^a. No cross-reaction between the shrew proteins and a rabbit anti-human Gc protein was observed.     

Horizontal polyacrylamide gradient gel electrophoresis for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle

A simple method of horizontal polyacrylamide gel electrophoresis was described for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle. A step gradient gel of 8, 4, 12 and 14 % acrylamide concentration was used. The method enabled the detection of a new protein polymorphism in the post-transferrin region. Two alleles were observed. The transferrin phenotypes involving D ₁ and D ₂ alleles were clearly separated. The resolution of the post-albumin fractions was also better than described by earlier methods.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles ( D,F,I,S ). Evidence was provided that the F allele can be further divided into two alleles ( F ₁ and F ₂); the mobilities of F₁ and F₂ variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Post-albumin, a foetal-specific rat plasma protein: Purification, physicochemical and immunological studies

1. The foetal-specific post-albumin of rat plasma was purified by electrophoresis on a Pevicon block. 2. The sedimentation coefficient, diffusion coefficient and molecular weight were determined for this protein and found to be similar to those of adult rat plasma albumin. 3. Foetal post-albumin and adult albumin were compared immunologically and shown, with rabbit antisera, to share no antigenic determinants, suggesting different genetic control of the production of each. 4. It is suggested that the disappearance of post-albumin in the newborn animal may result from the disappearance of haemopoietic tissue from the rat liver with advancing age.     

Characterization of the protein kinase activity in beet root plasma membranes

Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca²⁺ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca²⁺ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-³²P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca²⁺, while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca²⁺-stimulated phosphoprotein band at 64 kDa.     

Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression

A fall in circulating levels of cardiac survival factor insulin-like growth factor 1 (IGF-1) contributes to cardiac aging. To better understand the role of IGF-1 in cardiac aging, we examined the influence of cardiac IGF-1 overexpression on lifespan, cardiomyocyte intracellular Ca²⁺ homeostasis, protein damage, apoptosis and expression of pro- and anti-apoptotic proteins in young and old mice. Mouse survival rate was constructed by the Kaplan–Meier curve. Intracellular Ca²⁺ was evaluated by fura-2 fluorescence. Protein damage was determined by protein carbonyl formation. Apoptosis was assessed by caspase-8 expression, caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay. Pro- and anti-apoptotic proteins including Bax, p53, pp53, Bcl2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) were assessed by Western blot. Aging decreased plasma in IGF-1 levels, elevated myocyte resting intracellular Ca²⁺ levels, reduced electrically stimulated rise in intracellular Ca²⁺ and delayed intracellular Ca²⁺ decay associated with enhanced protein carbonyl formation, caspase-8 expression and caspase-3 activity in FVB mice, all of which with the exception of elevated resting intracellular Ca²⁺ were attenuated by IGF-1. Aging up-regulated expression of Bax, Bcl2 and ARC, down-regulated XIAP expression and did not affect p53, pp53 and Omi/HtrA2. The IGF-1 transgene attenuated or nullified aging-induced changes in Bax, Bcl2 and XIAP. Our data suggest a beneficial role for IGF-1 in aging-induced survival, cardiac intracellular Ca²⁺ homeostasis, protein damage and apoptosis possibly related to pro- and anti-apoptotic proteins.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

Purification and characterization of a calcium-regulated protein kinase from beet root (Beta vulgaris) plasma membranes

A calcium-regulated protein kinase (CRPK) associated with the plasma membrane of beet root cells was purified by deoxycholate extraction of plasma membrane proteins followed by chromatography in DEAE- and phenyl-Sepharose columns. This procedure rendered a purified CRPK preparation with a specific activity higher than 2.2 μmol mg^-1min^-1 using histone III-S as substrate. The molecular mass of CRPK, estimated by SDS-PAGE, was 58 kDa. Gel filtration and glycerol gradient centrifugation indicated that the native enzyme is a monomer. Phosphorylation of histone by CRPK displayed a broad pH optimum between 7.0 and 8.2, Syntide-2, a CaM-kinase II substrate, was phosphorylated much more efficiently than kemptide, Ac-MBP (4–14), CKII substrate or poly-(Glu, Tyr) 4:1. The kinetics of this enzyme can be described by a steady-state ordered mechanism in which four bound Ca²⁺ ions are required for the phosphorylation of the peptide substrate. After Ca²⁺ binding, the affinity of the enzyme for ATP was not affected while that for syntide-2 or histone decreased. Curves of kinase activity vs ATP at different syntide-2 concentrations indicated that the K_m values for ATP or syntide-2 increased with increasing cosubstrate concentrations. Similar curves of kinase activity vs ATP at different histone concentrations indicated that the apparent affinities of CRPK for ATP or histone were not affected by the cosubstrate concentration. The purified CRPK was not recognized by antibodies against soybean or Arabidopsis calcium-dependent protein kinase (CDPK). These results suggest that CRPK and CDPKs could be different enzymes, although they show some similar properties.     

Rapid Brain Uptake of Manganese(II) Across the Blood-Brain Barrier

Abstract: ⁵⁴Mn²⁺ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of ∼6 × 10^-5 ml/s/g for brain and ∼7 × 10^-3 ml/s/g for choroid plexus. Influx from physiologic saline was three- to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of ⁵⁴Mn²⁺ in rat plasma was ≤30%. Purified albumin, transferrin, and α₂-macroglobulin were each found to bind ⁵⁴Mn²⁺ significantly and to restrict brain ⁵⁴Mn²⁺ influx. The results demonstrate that ⁵⁴Mn²⁺ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn²⁺ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism.     

Induced Ca2+ uptake and callose synthesis in suspension-cultured cells of Catharanthus roseus are decreased by the protein phosphatase inhibitor okadaic acid

Subtoxic concentrations of the saponin digitonin. the polyene antibiotic amphotericin B and the bacterial phytotoxin syringomycin induce increased uptake of ⁴⁵Ca²⁺ into suspension-cultured plant cells and a rapid Ca²⁺-dependent defense response, callose synthesis. Both reactions were inhibited by preincubation of the cells with okadaic acid, a specific inhibitor of type 1 and type 2A protein phosphatases. These results suggest that Ca²⁺ uptake induced by the above agents does not occur due to unspecific perturbation of plasma membrane permeability but involves transport proteins which are controlled by protein phosphorylation/dephosphorylation. Phosphoproteins appear also to be involved in the regulation of callose synthesis, although it remains open whether this control is effected at the level of Ca²⁺ transport or at the 1,3-ß-glucan synthase involved in deposition of the polymer.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

Dose-related effects of 17βoestradiol (E2) on liver weight, plasma E2, protein, calcium and thyroid hormone levels, and measurement of the binding of thyroid hormones to vitellogenin in rainbow trout, Salmo gairdneri Richardson

Administration of 17β-oestradiol (E₂) to rainbow trout, in the form of hydrogenated coconut oil implants produced a stable, long-term elevation in plasma E₂ levels. The elevation was doserelated (over the range 1–10mg kg-¹ body weight) both 4 and 8 weeks after implantation. Dose-related increases were also observed with respect to liver weight-body weight ratios and plasma protein levels. Plasma T₃ and total calcium levels were depressed and elevated, respectively, by E₂ treatment but the responses were not linearly related to the dose of E₂ administered; there was no significant effect of E₂ on plasma T₄ levels.
E₂ induced a shift in the binding of T₃ to plasma proteins, with T₃ binding to smaller molecular weight proteins; neither T₄ nor T₃ bound to vitellogenin which was present at high levels in the plasma of E₂-treated fish.     

Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo , there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrP^C) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrP^C as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrP^C, but they do appreciably modify the accessibility of the polypeptide surface PrP^C. In addition, the simulations of membrane-bound PrP^C revealed likely recognition domains for the disease-initiating PrP^C:PrP^Sc (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species.     

Neuroendocrine stimulation of uterine gland protein synthesis in the tsetse fly, Glossina morsitans

ABSTRACT. The uterine gland of the tsetse fly Glossina morsitans morsitans Westw. synthesizes a secretion which nourishes the developing larva in the uterus. Aqueous extracts of the brain have been shown to stimulate the synthesis of the protein and amino acid components of this secretion from L- [U-¹⁴C]leucine by uterine gland tubules in vivo and in vitro. A linear dose response relationship was demonstrated in vitro with extract concentrations ranging from 1 × 10^-4 to 1 × 10^-2 brains μl^-1. The maximum response, a > 300% increase in the rate of protein and amino acid synthesis, was achieved with as little as 1 × 10^-2 brains μl^-1 The concentration of active factor(s) in the brain declined during a single interlarval period coincident with the period of release of secretion associated with larval growth. The stimulatory activity in brain extracts was destroyed by proteolytic enzymes indicating that it is probably a protein or peptide. Results suggest that the active factor(s) is a hormone responsible for the stimulation of uterine gland protein synthesis essential for larval nutrition.     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

Protein turnover is generally regarded as a major maintenance process, but experimental evidence to support this contention is scarce. Here we quantify the component of dark respiration rate associated with overall protein turnover of tissues in vivo. The effect of an inhibitor of cytosolic protein synthesis (cycloheximide, CHM) on dark respiration was tested on a cell suspension from potato ( Solanum tuberosum L.) and quantified on leaf discs of expanding and full-grown primary leaves of bean ( Phaseolus vulgaris L.). The in vivo effect of CHM on protein biosynthesis was assessed by monitoring the inhibition of the induction of the ethylene-forming enzyme (EFE) activity. The present method yields the energy costs of turnover of the total pool of proteins irrespective of their individual turnover rates. Average turnover rates were derived from the respiratory costs and the specific costs for turnover.
Inhibition of respiration by CHM was readily detectable in growing-cell suspensions and discs of expanding leaves, The derived respiratory costs of protein turnover in expanding leaves were maximally 17–37% of total respiration. Turnover costs in full-grown primary leaves of bean amounted to 17–21% of total dark respiration. The maximum degradation constants (i.e. K_d-values) derived for growing and full-grown leaves were up to 2.42 × 10⁻⁶ and 1.12 × l0⁻⁶ s⁻¹, respectively.