α-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide

alpha-Crystallin was carboxymethylated with radioactive iodoacetic acid in the presence of 7.6m-urea and then separated into six major fractions by chromatography on DEAE-cellulose in 7m-urea. Based on the amino acid compositions, specific radioactivities and sodium dodecyl sulphate-gel electrophoresis of the fractions, it was concluded that alpha-crystallin contains at least four different subunits: DU1A and DU1B, containing no cysteine; a third component represented by DU2B and DU3 containing one cysteine one cysteine residue per subunit; and DU4, which probably contains two residues of cysteine per subunit. Subunit DU1A was shown to be of sufficient purity for sequence studies. Cyanogen bromide cleavage yielded two peptides, CB-1 and CB-2, in approximately equal amounts as expected. The sum of the molecular weights and amino acid compositions of the peptides were both in excellent agreement with the results obtained for subunit DU1A. The amino acid sequence of the first sixteen residues of peptide CB-1 is: Ser-Leu-Thr-Lys-Asp-Phe-Asp-Glu-Val-Asn-Ile-Asp-Val-Ser-His-Phe-. The sequence of the first seventeen residues of peptide CB-2 is: Asp-Ile-Ala-Ile-Ser-His-Pro-Trp-Ile-Arg-Pro-Ser-Phe-Phe-Glu-Phe-His-. The N-terminal sequence of subunit DU1A was shown to be N-acetylmethionine followed by peptide CB-2.     

Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase

The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.     

Characterization of a monoclonal antibody which is an activator of rabbit platelets

A monoclonal antibody (MAb) raised against rabbit platelet membranes was shown to be a strong agonist to induce platelet aggregation and secretion. This MAb, designated 19CB-1, was identified as an IgM and purified to near homogeneity by ammonium sulfate precipitation and Q-sepharose column chromatography. Aggregation induced by 19CB-1 was only slightly affected in the presence of creatine phosphate/creatine phosphokinase and aspirin, indicating that it was not mediated through the cyclooxygenase pathway and the release of ADP. 19CB-1 Fab fragments did not induce platelet aggregation. However, 19CB-1-induced aggregation was inhibited by these Fab fragments. 19CB-1 also elicited a rise in cytoplasmic calcium concentration in fura-2 loaded platelets. In the absence of external calcium, a substantial calcium signal remained to be observed, suggesting the release of calcium from intracellular stores in response to 19CB-1. This MAb reacted primarily with a polypeptide of Mr = 57,000, as revealed by immunoblotting. These results suggest that the 57 kDa antigen is one of the platelet surface proteins directly involved in the activation of rabbit platelets.     

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I

A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.     

Serum prolactin levels of rats under continuous estrogen stimulation and 2 Br-alpha-ergocryptine (CB-154) injection.

The ability of 2 Br-alpha-ergocryptine (CB-154) to suppress serum prolactin levels was examined in animals under the influence of continuous estrogen stimulation. A single injection of polyestradiol phosphate (Estradurin) once every 21 days produced a constant elevation of serum prolactin. The simultaneous administration of 1 mg/day of CB-154 to estrogenized animals suppressed serum prolactin levels below that of Estradurin alone but the levels were significantly greater than those of animals receiving CB-154 alone. It was suggested that, while CB-154 prevents the release of prolactin, estrogen stimulates prolactin synthesis despite the block of its release. Ether anesthesia may be capable of partially overriding the block of CB-154 and released the stored hormone from the gland.     

Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.     

Preliminary studies on the primary structure of rat liver dihydropteridine reductase

Cyanogen bromide cleavage of reductively alkylated homogeneous rat liver dihydropteridine reductase afforded several peptide fragments identifiable by polyacrylamide electrophoresis of which 6 (CB-1 to CB-6) could be individually isolated by C8 reverse phase HPLC. Each was characterised by N-terminal amino acid analysis and sequence information was derived for CB-1, CB-4 and CB-6. The blocked N-terminal of the holoenzyme was identified as pyroglutamate and the C-terminal sequence was obtained by sequential degradation.     

Prolactin modulation of the maternal-like behavior displayed by juvenile rats

Reports of elevated prolactin (Prl) levels in juvenile rats of the same strain and approximate age, together with the established role of Prl in maternal behavior in adult female rats, prompted us to examine the possible involvement of Prl in the expression of maternal-like behavior in juvenile Sprague-Dawley males and females. Experiment 1 showed that at 25 days of age both sexes exhibited a rapid onset of full maternal behavior (FMB), with males (median = 2.0 days) responding significantly more quickly than females (median = 4.0 days). Moreover, blood sampled for Prl revealed that males had significantly higher levels of circulating Prl than females, (21.0 vs 10.4 ng/ml, respectively). In Experiment 2, CB-154 treatment significantly delayed the onset of FMB in males only, causing latencies to increase to 5.0 days vs 2.0 days for Controls. Female latencies were unaffected by CB-154, 7.0 and 7.5 days for CB-154 and Control groups, respectively. A second set of both male and female juveniles was treated with either CB-154 or vehicle. CB-154 reduced Prl levels in both sexes. In the Controls, the sex difference in Prl levels (males greater than females) was again evident. In Experiment 3 juvenile males were treated with either ovine Prl (0.5 mg) + CB-154, CB-154 + Vehicle, or Vehicle + Vehicle and tested for FMB. Males treated with Prl + CB-154 required 3.0 days to exhibit FMB, significantly faster than CB-154 + Vehicle males which responded in 8.0 days. The response of Vehicle + Vehicle males was intermediate, with a latency of 5.0 days. These results provide support for the idea that Prl is involved in the maternal-like responsiveness shown by 25 day old juvenile males, but that in females a maturational factor may have prevented both heightened responsiveness to pups by 25 days of age and sensitivity to the Prl releasing mechanism(s)/Prl feedback involved in the exhibition of maternal behavior.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

The primary structure of actin from rabbit skeletal muscle. Five cyanogen bromide peptides, including the NH2 and COOH termini.

As a part of a study of the complete amino acid sequence of actin we have determined the sequences of five cyanogen bromide peptides, which together contain 158 amino acid residues, including the two ends of the molecule. The five peptides are: CB-13 (residues 1 to 44 in the intact chain), CB-11 (residues 83 to 119), CB-12 (residues 228 to 268), CB-8 (residues 283 to 298), and CB-9 (residues 355 to 374). Each of the peptides except CB-11 has one sulfhydryl group, and these peptides thus account for 4 of the 5 cysteines in actin. The reactivity of actin --SH groups toward N-ethylmaleimide was investigated, and it was found that Cys-373 (in CB-9 adjacent to the COOH-terminal phenylalanine) is the first to react with this reagent.     

Effects of in vivo and in vitro exposure to bromocriptine on testicular LH receptors and in vitro testosterone production in Syrian hamsters.

The effects of in vivo and in vitro exposure to bromocriptine (CB-154) were studied in testes of Syrian hamsters. In animals treated for two days with CB-154, a decrease in LH receptors (LH-R) was observed, with a greater decrease being measured in hamsters treated for 14 days, when compared with controls. Injection of HCG caused, in hamsters treated with CB-154 for 14 days, up-regulation of LH-R and increased testosterone synthesis in response to HCG administration in vitro. These changes were not observed in the two other groups of animals. When testis fragments were incubated with CB-154, those incubated with a large dose (10 micrograms/ml) had a normal pattern of response to HCG, and those incubated with a small dose (1 ng/ml) had a smaller maximum response. These actions are similar to those observed in men treated with CB-154. It can be therefore concluded that: a) CB-154 has a direct effect on the testes; b) it probably is through modulation of LH-R synthesis; c) Syrian hamsters probably represent the best model for the study of the effects of CB-154 on the testes; and d) the possibility of using CB-154 as an adjuvant of gonadotropin treatment in hypogonadism has to be considered.     

Studies on the alkali light chains of vertebrate skeletal muscle myosin. Effect of tyrosyl modification on the ability to reassociate to heavy chains

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr-cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB-1 and CB-3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB-6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109, 455-470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908-5913]. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB-1 and CB-3 are involved in the association to the heavy chains.     

Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water

Several poliovirus and coxsackievirus isolates from environmental sources were compared with laboratory strains to determine their rate of inactivation by chlorine. All viruses were tested for up to 1,000 min in the presence of an initial free residual chlorine level of ca. 0.4 mg/liter. Coxsackievirus B5 (CB-5) isolates were found to be more resistant to chlorine than coxsackievirus B4 (CB-4), followed by poliovirus 1, 2, and 3 in order of decreasing resistance to chlorine. Environmental isolates of CB-5 were more resistant than the laboratory strain tested, and for two strains 12 and 22% of the input virus was still infectious after 100 min in the presence of free residual chlorine. Although CB-4 isolates were less resistant to chlorine than CB-5 isolates, after 1,000 min of contact 0.01% of the input virus was still infectious. Except for CB-5 isolates, isolates from environmental sources did not appear to be more resistant to chlorine than laboratory strains. Viruses isolated at different phases during the preparation of drinking water were not more resistant to chlorine and must thus have been protected by other mechanisms.     

Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water.

Several poliovirus and coxsackievirus isolates from environmental sources were compared with laboratory strains to determine their rate of inactivation by chlorine. All viruses were tested for up to 1,000 min in the presence of an initial free residual chlorine level of ca. 0.4 mg/liter. Coxsackievirus B5 (CB-5) isolates were found to be more resistant to chlorine than coxsackievirus B4 (CB-4), followed by poliovirus 1, 2, and 3 in order of decreasing resistance to chlorine. Environmental isolates of CB-5 were more resistant than the laboratory strain tested, and for two strains 12 and 22% of the input virus was still infectious after 100 min in the presence of free residual chlorine. Although CB-4 isolates were less resistant to chlorine than CB-5 isolates, after 1,000 min of contact 0.01% of the input virus was still infectious. Except for CB-5 isolates, isolates from environmental sources did not appear to be more resistant to chlorine than laboratory strains. Viruses isolated at different phases during the preparation of drinking water were not more resistant to chlorine and must thus have been protected by other mechanisms.     

Effect of bromocryptine on the secretion of thyrotropic hormone (TSH), prolactin (Pr), human growth hormone (HGH), thyroxine (T4) and triiodothyroxine (T3) in hypothyroidism.

Bromocryptine (CB-154) virtually abolished the rise of serum Pr after TRH stimulation in hypothyroid and euthyroid subjects. The response of serum TSH to TRH stimulation was significantly depressed in hypothyroid but not in euthyroid subjects. No significant changes of serum HGH, T4 and T3 after CB-154 were observed. The dual mode of action of CB-154 in pituitary and hypothalamus is discussed.     

Activation of glycogen synthase by insulin in rat adipocytes. Evidence of hormonal stimulation of multisite dephosphorylation by glucose transport-dependent and -independent pathways

Adipocytes were incubated with [32P]phosphate to achieve steady state labeling of glycogen synthase. The enzyme was then rapidly immunoprecipitated and subjected to electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The 32P-labeled glycogen synthase had an apparent molecular weight ( Mapp ) equal to 90,000. All of the [32P]phosphate could be recovered in two cyanogen bromide fragments. The larger fragment, CB-2 ( Mapp = 28,000), contained about five times more [32P]phosphate than the smaller fragment, CB-1 ( Mapp = 15,500). Insulin increased the activity ratio (-glucose-6-P/+glucose-6-P) of glycogen synthase from 0.12 to 0.26, but did not decrease the amount of [32P]phosphate in the enzyme. However, insulin promoted the formation of species of CB-2 of lower Mapp , suggesting dephosphorylation of sites that affected the electrophoretic mobility of the fragment. Glucose did not affect the mobility of CB-2, but slightly increased the activity ratio and decreased the [32P] phosphate by approximately 20%. With insulin plus glucose, the increase in activity ratio was much greater than the additive effects of either agent alone. The combination decreased the [32P]phosphate in each cyanogen bromide fragment by approximately 60%, indicating that the synergistic activation was due to enhanced dephosphorylation of multiple sites. 2-Deoxyglucose also promoted dephosphorylation of glycogen synthase, decreasing the 32P content of CB-1 and CB-2 by approximately 40% each. 3-O-Methylglucose was without effect. The results presented suggest that the activation of glycogen synthase by insulin via a glucose transport-dependent pathway may involve increased intracellular glucose-6-P which promotes dephosphorylation of sites in both CB-1 and CB-2. Activation by a glucose transport-independent pathway appears to be confined to sites located in CB-2.     

Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism

We have studied the expression of Ia molecules by macrophages from mice with severe combined immunodeficiency (CB-17 scid) that lack demonstrable T cell and B cell functions. CB-17 scid mice had approximately normal numbers of Ia-bearing macrophages in the peritoneal cavity, spleen, and liver. Peritoneal macrophages responded in culture to T cell-derived lymphokines with enhanced expression of Ia molecules. However, unlike immunocompetent controls, SCID mice could not enhance Ia expression in an antigen-specific T cell-dependent manner after secondary challenge in vivo with a conventional protein antigen such as hemocyanin. Further demonstration of their T cell deficiency was the failure of CB-17 scid spleen cells to proliferate and produce IL 2 in response to the T cell mitogen, concanavalin A. Upon infection with Listeria monocytogenes, CB-17 scid mice developed chronically high loads of bacteria, whereas CB-17 control mice eliminated all viable bacteria and became resistant to secondary infection. However, Listeria-infected CB-17 scid mice did show, in parallel with the CB-17 controls, an unexpected and striking increase of Ia-positive macrophages. These data indicate that induction of Ia expression in macrophages can occur via a mechanism that is independent of mature T cells.     

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium

A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO₂. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Received: 6 November 1998 / Accepted: 19 January 1999     

Phosphorylation and inactivation of rat hepatocyte glycogen synthase by phorbol esters and mezerein

Incubation of rat hepatocytes with active phorbol esters and mezerein provoked a decrease in glycogen synthase activity. After the incubation of [3 2 P] phosphate-labeled cells with these tumor promoters, an increase in the amount of 3 2 P bound to the immunoprecipitated enzyme was observed. The decrease in activity highly correlated with the phosphorylation in the smaller CNBr fragment (CB-1) and only at high concentration of the phorbol ester the increase in the phosphorylation of the larger CNBr fragment (CB-2) became significative. Tryptic degradation of CB-1 showed two phosphopeptides after isoelectro focusing analysis (pI 3.9 and pI 3.4) and only one of them (pI 3.9) increased its phosphorylation state after treatment of the cells. These results indicate that the decrease in activity of glycogen synthase by phorbol esters and mezerein is a result of the phosphorylation of the enzyme and that a single site located in CB-1 is preferentially phosphorylated by these agents.