Electron spin resonance studies on the conformation and interactions of histories containing cysteine. Histones H3 from calf thymus and H4 from sea urchin sperm.

In this study the spin-label method has been used to obtain information about conformational properties of regions containing cysteine of histone H3 from calf thymus, histone H4 from sperm of the sea urchin Arbacia lixula, and the histone complex H3–H4. It has been found that the microenvironments of histone H3 causing immobilization of the spin labels are sensitive to variations in ionic strength of dilute solutions of phosphate buffer, are partially destroyed by urea, and fully destroyed by proteolytic enzymes. The interaction of spin-labeled histone H3 with histone H4 induces an increase of immobilization of the spin label, indicating an increase in rigidity at the cysteine region of histone H3. The use of a series of spin labels of variable length for histone H3 gives an estimate of 0.8–1.0 nm for the apparent depth of the spin label binding site, a value which does not change upon interaction of histone H3 with H4. Histone H4 from A. lixula sperm causes a similar immobilization of the spin label. As for histone H3, immobilization increases with the ionic strength, and the structures are destroyed by urea and proteolytic enzymes. Upon mixing with histone H3, however, the extent of immobilization appears only slightly changed, and together with sedimentation velocity results, these studies suggest that the spin label attached to histone H4 prevents the complex formation.     

Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.     

Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus

Phosphorylation of histone H1 occurs when spermatozoa of the sea urchin Strongylocentrotus purpuratus are treated with the macromolecular fraction of solubilized egg jelly. Phosphorylation is on serine residues in the N-terminal fragment of H1 bisected with N-bromosuccinimide. Phosphorylation is maximal by 4-8 min and dependent on Ca2+, but independent of Na+ or increased intracellular pH. Phosphorylation of H1 can be dissociated from the induction of the acrosome reaction. Only a fraction of the H1 molecules become phosphorylated upon treatment of sperm with egg jelly. The amount of phosphate per mole of H1 increases from 0.15 moles before jelly treatment to 0.46 moles after maximal phosphorylation. Phosphorylation of H1 occurs in a cAMP-dependent manner as indicated by the ability of the phosphodiesterase inhibitors IBMX and SQ20009 to induce H1 phosphorylation. This phosphorylation reaction can be blocked by digesting the sperm surface with Pronase, or preincubation of sperm in wheat germ agglutinin, showing that a ligand in egg jelly must interact with a sperm surface receptor to activate the kinase phosphorylating H1.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

A Zn2+-dependent and histone H1-specific protease in sea urchin sperm

Protease activity was extracted from sea urchin sperm with 1% Triton X-100 and partially purified by DEAE-cellulose and Sephadex G-100 chromatography. The enzyme preferentially degraded histone H1, while showing only a weak activity toward other histones. Heat-denatured casein and bovine serum albumin were not digested by this enzyme under the present experimental conditions. This protease hydrolyzed only Boc-Val-Leu-Lys-MCA among various peptidyl-MCAs. The optimal pH ranged from 7 to 11. Its molecular weight was about 41,000. Among various known inhibitors of proteases, only omicron-phenanthroline effectively inhibited the activity. The enzyme was stimulated by Zn2+ or Co2+. It was inactivated by omicron-phenanthroline but could be reactivated by the addition of Zn2+ or Co2+. Therefore, this protease seems to be a metalloprotease dependent on Zn2+ or Co2+. The insensitivity of this enzyme to phosphoramidon and its very restricted substrate specificity suggest that this enzyme is very different from other metalloproteases described hitherto.     

Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

A stable alpha-helical element in the carboxy-terminal domain of free and chromatin-bound histone H1 from sea urchin sperm.

The carboxy-terminal domain (residues 121-248) of sea urchin sperm-specific H1 is not random coil but partly alpha-helical, even in 1 mM sodium phosphate, pH 7. The helix resides in a 57 residue proline-free segment which, in the intact histone, immediately abuts the central globular domain. The proline-free region, which is rich in lysine and alanine, is relatively resistant to tryptic digestion when the carboxy-terminal domain is bound to DNA. Two (overlapping) resistant peptides are shown by circular dichroism measurements to be substantially alpha-helical in 1 mM sodium phosphate and to increase in helix content to approximately 70% in 1 M NaCLO4. Tryptic digestion of chromatin gives resistant fragments containing both the globular domain and the contiguous proline-free segment, strongly suggesting that the alpha-helical segment also exists in chromatin, where it would be ideally placed to direct the path of the linker DNA entering or leaving the nucleosome. The linker in sea urchin sperm chromatin is long (approximately 74 bp), and the unusually long alpha-helical segment in the carboxy-terminal tail of sperm H1 which has amphipathic character due to the alanine distribution, and is likely to be curved, may be a special feature tailored to organize it.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

The amino terminal sequence of sea urchin sperm histone H1 and its phosphorylation by egg cytosol

1. The amino acid sequence of the first 34 residues of sperm histone H1 (SpH1) from Strongylocentrotus purpuratus shows striking similarity with sequences from three South African species. 2. Five contiguous repeats of the tetrapeptide SPBB (where B is R or K) occur between positions 10 and 29. 3. SpH1 was phosphorylated in vitro using egg cytosol as the source of protein kinase and approximately 4.2 mol phosphate were incorporated per mol H1. 4. Sequences of five phosphopeptides of SpH1 show the egg possesses protein kinase activity capable of phosphorylating multiple seryl residues including SPBB in the NH2-, and BBSP in the COOH-end of the protein.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.     

Evolution of late H2A, H2B, and H4 histone genes of the sea urchin, Strongylocentrotus purpuratus.

Sea urchins possess several distinct sets of histone genes, including "early" genes, maximally active in cleavage and blastula stages, and "late" genes, active from the late blastula stage onwards. We determined the nucleotide sequences of six sea urchin (Strongylocentrotus purpuratus) late histone genes located on four genomic segments. Comparative analysis of these sequences identified several conserved elements in 5' flanking regions, including the sequences ATGPyATANTATA shared by all late genes and GGCGGGAAATTGAAAA shared by two late H4s. Comparisons of protein-coding sequences of late H4 and H2B genes with their early counterparts showed that silent sites have diverged to the theoretical maximum, indicating that early and late histone gene classes diverged at least 200 million years ago. Since extant echinoderms evolved from a common ancestor at about that time, it is likely that early and late histone gene sets are characteristic of all echinoderm groups. Amino acid sequences derived from nucleotide sequences of late H2A and H2B gistone genes differ substantially from amino acid sequences of their late counterparts. Most such differences are in highly mutable positions. A few, however, occur in positions that do not mutate frequently and thus may reflect functional differences between the early and late forms of the H2A and H2B proteins.     

Isolation and some properties of Ca2+-, Mg2+-dependent deoxyribonuclease from sea urchin (Strongylocentrotus intermedius) embryos]

Ca2+-Mg2+-dependent deoxyribonuclease (deoxyribonucleate-5'-oligonucleotidehydrolase E. C. 3.1.4.5). Molecular weight of the enzyme is found to be 40 000 daltons isoelectric point--4.4. The enzyme degraded DNA only in the presence of bivalent cations. It hydrolyses preferentially native DNA with pH optimum 7.0-7.2 in the presence of Mg2+ ions. Ca2+ ions shift the pH optimum to 8.0-8.5. Combined addition of Ca2+ and Mg2+ ions results in a sinergic effect and changes the enzyme specificity to the secondary DNA structure. The enzyme hydrolyses both native and denatured DNA by the endonucleolytic type to form oligonucleotides with 5' terminal phosphate the content of tetra-octanucleotides being 80-85%.     

Characterization of the structure and transcriptional patterns of the gene encoding the late histone subtype H1-beta of the sea urchin Strongylocentrotus purpuratus.

We have cloned and characterized the gene encoding the late histone H1-beta subtype from the sea urchin Strongylocentrotus purpuratus. The gene contains all of the upstream sequence homologies previously seen in late H1-gamma genes. The expression of H1-beta mRNA is coordinated with that of H1-gamma mRNA, and like H1-gamma it is expressed in all adult somatic tissues tested.     

Cysteine-containing histone H1-like (PL-I) proteins of sperm.

We have determined the presence of cysteine in the protein PL-I from the sperm of the surf clam Spisula solidissima. The existence of cysteine in this histone H1-related protein is responsible for its previously described aggregation behavior. The location of this residue, within the trypsin-resistant domain of the protein, has been established. We have also shown that cysteine is ubiquitously present in the PL-I proteins from the sperm of other bivalve mollusks but is absent from other PL of smaller molecular mass (PL-II, PL-III, PL-IV). We have also found cysteine to be present in the PL-I from a tunicate (Chelysoma productum) but absent in a PL-I from a fish (Mullus barbatus). The possible significance of the unusual occurrence of cysteine in these histone-H1-related proteins is discussed.