Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation

We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.     

The majority of potassium ions in muscle cells is adsorbed on beta- and gamma-carboxyl groups of myosin: potassium-ion-adsorbing carboxyl groups on myosin heads engage in cross-bridge formation during contraction.

High-molecular weight poly(ethylene glycol) (PEG-8000) in the bathing medium prolongs the survival of 2-mm-wide frog muscle segments with open ends. In a PEG-8000-containing medium Rb+, K+, and Na+ in the muscle segments reached new diffusion equilibrium in 2-4 hours. At this new equilibrium, the cell's preference of K+ over Na+ was preserved but very much weakened. Studies of the influence of pH on the equilibrium distribution of labelled Na+ in 2-mm-wide muscle segments confirmed the prediction that beta- and gamma-carboxyl groups, carried respectively on aspartic and glutamic acid residues of intracellular proteins, adsorb K+, Na+ and other monovalent cations. These carboxyl groups have a characteristic pKa between 3.65 and 4.25. A pKa of 3.85 was observed. These findings, when seen in the light of other relevant information available, led to the conclusion that beta- and gamma-carboxyl groups on myosin molecules adsorb--in a close contact one-ion-one-site fashion--the majority (67% to 80%) of K+ in resting muscle cells. Other evidence suggests that in muscle contraction, the K(+)-adsorbing beta- and gamma-carboxyl groups on myosin heads form salt linkages with cationic sites on actin, displacing and releasing the adsorbed K+. Present and earlier findings together offer support for an earlier suggestion that the formation and dissociation of these salt-linkages may underlie the force-generating, cyclic formation and dissociation of cross-bridges during muscle contraction.     

Modulation of hepatic level of microsomal testosterone 7 alpha-hydroxylase, P-450a (P450IIA), by thyroid hormone and growth hormone in rat liver

The effects of thyroid hormone and growth hormone on microsomal testosterone 7 alpha-hydroxylase, P-450a, were studied to understand the interaction of these hormone-mediated regulations in rats. In Western blots using anti-P-450a IgG, 1.7-fold higher content of P-450a was observed in livers of female than male adult rats, while no appreciable sex-related difference was detected in prepubertal rats and rats of 24 months of age. Treatment with n-propyl-2-thiouracil or thyroidectomy of male rats increased by 2-fold the hepatic content of P-450a, but neither regimen had a significant effect on the content in female rats. Levels of P-450a in both sexes of thyroidectomized rats were decreased by the supplementation of triiodothyronine (T3, 50 micrograms per kg, i.p. for 7 days) to levels similar to that observed in normal male rats. Hypophysectomy also caused an increase in microsomal P-450a content in male rats. Continuous infusion of human growth hormone, which mimicked the female secretion, further significantly increased the content in hypophysectomized rats to a level similar to that observed in normal female rats. In contrast, hepatic level of P-450a in hypophysectomized male and female rats was reduced by intermittent injection, which mimicked the male secretion. Clear suppression on the level of hepatic P-450a was also observed by the treatment of hypophysectomized rats with 5 or 50 micrograms/kg of T3 and of hGH-infused hypophysectomized rat with 50 micrograms/kg of T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II)

A series of murine thymic lymphoma cell sublines was selected in vitro for resistance to cis-diamminedichloroplatinum(II) (CDDP). The level of CDDP resistance correlated with reduced drug accumulation in these cells. A rabbit antiserum was raised against the plasma membrane of a CDDP-resistant subline and used in Western blot analyses. Increased expression of a surface antigen of approximately 200 kDa was observed and found to correlate with the degree of resistance. Further biochemical and immunological studies demonstrated that this is a plasma membrane glycoprotein. However, it is different from the multidrug resistance-associated P-glycoprotein with a molecular weight of about 170,000. We have called this unique CDDP resistance-associated membrane protein CPR-200.     

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.     

Genetic instability of R plasmids in relation to the shift of drug resistance patterns in Salmonella johannesburg

Observation of the resistance of Salmonella johannesburg to the six drugs ampicillin (A), streptomycin (S), tetracycline (T), chloramphenicol (C), kanamycin(K) and sulphadiazine (Su) was made over the 7 years from 1973 to 1979. Strains with ASTCKSu- and ASCKSu- resistance patterns predominated in the years 1973-1975 and 1976-1979, respectively. These resistances were found to be mediated by autotransferring plasmids belonging to the incompatibility group FIme. The ASTCKSu-resistance plasmids were unstable, giving rise to deletion variants at a much higher frequency than ASCKSu-resistance plasmids either of natural origin or derived in vitro from the ASTCKSu-resistance plasmids. Thus, the ASCKSu-resistance plasmid might be a deletion variant of the ASTCKSu-resistance plasmid. This is supported by the extensive similarity of their cleavage patterns produced by specific restriction endonucleases.     

Genetic specificity of DNA synthesized in the absence of T4 bacteriophage gene 44 protein.

Upon infection of Escherichia coli B with T4 phage with DO amber mutation in gene 44, a minimal amount of phage DNA is synthesized. This progeny DNA is, for the most part, covalently attached to the parental DNA. Analysis of the genetic representation of this DNA was performed by hybridization to cloned genetic segments. It was shown that areas preferentially replicated differ from origins observed in "normal" replication: under normal conditions, there is a strong origin in the genetic area of genes 50-5 and lack of initiation within the group of genes 40-43 and 35-52. In contrast, in the absence of the gene 44 protein, the genetic area of 50-5 is underrepresented, genes 35-36, tRNA, and genes 40-41 are the most prominent among progeny DNA, and the area of gene 39 is least represented. Since the area of gene 35 is known from the genetic data or other to be a high-frequency recombination area, and since the area of gene 39 is known to display a low frequency of recombination, we postulate that the observed uptake of label occurs at the site-specific recombinational intersections.     

Synthesis of a glycotripeptide and a glycosomatostatin containing the 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-serine residue

3-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-N- (tert-butyloxycarbonyl)-L-serine was synthesized and condensed by the solid-phase procedure to give the sequence Gly-[beta-D-GlcpNAc-(1 leads to 3)-Ser]-Ala-OH and beta-D-GlcpNAc-(1 leads to 3)-Ser-13-somatostatin. The synthetic glycopeptides appeared homogeneous on t.l.c. and l.c. examination and showed the correct amino acid composition and 2-amino-2-deoxy-D-glucose content. The structure of Gly-[beta-D-GlcpNAc-(1 leads to 3)-Ser]-Ala-OH was further confirmed by mass spectrometry of the N-acetyl permethyl derivative, and by n.m.r. spectroscopy.     

Solid phase synthesis of somatostatin-28

The synthesis of ovine hypothalamic somatostatin-28 (Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-$\text{Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys}$-OH) has been accomplished by solid phase methodology. The structure of the synthetic material was verified by: (1) direct sequence analysis with a Beckman 89°C sequencer, (2) correlation of the amino acid analyses of the isolated tryptic peptide fragments with their theoretical compositions, and (3) comparison, using high performance liquid chromatography, of the synthetic methionine-sulfoxide and methionine-sulfone modified NH₂-terminal peptides (residues 1–11) with the corresponding tryptic fragment from somatostatin-28.     

Synthesis and biological activity of glycosylated analogs of somatostatin

The synthesis by solid-phase methodology of two glycosylated analogs of somatostatin [Glc-Asn⁵]-SS and [NAcGlc-Asn⁵]-SS is described. These two analogs have been biologically tested on the secretion of pituitary growth hormone, pancreatic glucagon and insulin. The results show that glycosylation of somatostatin on the Asn⁵ residue decreases by a hundred fold the inhibition activity on GH release when tested $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$, since the activity is similar to somatostatin the carbohydrates are probably removed by some enzymatic reaction and thus liberate the full activity of somatostatin.