M. Gruber

Structure and properties of hemocyanins: I. Electron micrographs of hemocyanin and apohemocyanin from Helix pomatia at different pH values

Electron micrographs were made from Helix pomatia hemocyanin at different pH values with the negative-staining method. The pH-dependent dissociation on the alkaline side of the isoelectric point was especially studied. Our observations are in agreement with previously reported results, although they are not compatible with the model and mechanism of dissociation previously proposed. Our observations suggest that the Helix pomatia hemocyanin molecule is roughly cylindrical ; the diameter of the cylinder is about 300 A and the height is about 335 A. This cylinder has a fivefold axis; perpendicular to this axis it is built up from 6 parallel layers. The first dissociation step occurs perpendicular to the fivefold axis into halves. Further dissociation is supposed to be a splitting of these flat cylinders via submultiples into subunits. Divalent cations in concentrations of about 10 −4 molar or higher are required for the reversibility of this dissociation. Helix pomatia apohemocyanin shows a similar behaviour.

Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein

The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.

Role of individual cathepsins in lysosomal protein digestion as tested by specific inhibitors

Abstract We have investigated the effect of some inhibitors on the breakdown of proteins at 38 °C and pH 5 by highly purified Triton WR-1339-filled lysosomes obtained from rat liver. When cathepsin D (EC 3.4.23.5) was completely inhibited by pepstatin, the rate of degradation of native serum albumin was not and that of carboxymethylated serum albumin only slightly influenced. Leupeptin or antipain, used at a concentration which resulted in more than 95% inhibition of cathepsin B1 (EC 3.4.22.1) activity, strongly decreased the rate of degradation of native albumin, but affected the degradation of carboxymethylated albumin or hemoglobin only to a slight degree. Leupeptin or antipain in combination with pepstatin inhibited proteolysis of the three proteins strongly, but not completely. Omission of Cl − , essential for cathepsin C (EC 3.4.14.1) activity, from incubation mixtures containing pepstatin, leupeptin and antipain had only a slight additional inhibitory effect. Addition of both pepstatin and monoiodoacetate fully suppressed the degradation of these protein substrates. Some of the experiments have also been done using Triton WR-1339-filled lysosomes obtained from purified hepatocytes. Our results indicate that (1) cathepsin D is not essential for degradation of proteins by liver lysosomal enzymes: (2) cathepsin B1 (or possibly another leupeptin-inhibited thiol protease) is the most important enzyme in the degradation of native albumin; (3) thiol enzymes, other than cathepsin B1, C or D, are involved in lysosomal protein degradation; (4) hepatocyte lysosomes contain a set of proteases sufficient for rapid and extensive degradation of albumin.

Structure and properties of hemocyanins. V. Binding of oxygen and copper in Helix pomatia hemocyanin

Abstract 1. 1. Reintroduction of monovalent copper into apo-α-hemocyanin of Helix pomatia leads to the complete return of the properties of native α-hemocyanin. 2. 2. The copper atoms in hemocyanin are required for the reassociation of tenth and twentieth molecules. 3. 3. In the reintroduction process, the copper atoms are randomly distributed among the empty binding sites of apo-α-hemocyanin. All copper-binding sites are equivalent and grouped in pairs; each pair forms a functional group. 4. 4. Upon storage for a few months, part of the copper atoms in every hemocyanin molecule are oxidized to Cu 2+ . This affects the oxygenation properties. 5. 5. The oxygenation curves of fresh α- and β-hemocyanin are nonhyperbolic at alkaline pH values in the presence of Ca 2+ and Mg 2+ . The influence of the pH on the oxygenation curves of α- and β-hemocyanin shows marked differences.

Structure and properties of hemocyanins VI. Association-dissociation behavior of Helix pomatia hemocyanin

Abstract 1. 1. The association-dissociation behavior of α-hemocyanin of Helix pomatia was studied by ultracentrifugal analysis. 2. 2. Above pH 7 and below pH 5 whole molecules (102 S) dissociate into half (64 S) and tenth (20 S) molecules. At alkaline pH further dissociation is observed into twentieth (13 S) molecules (mol.wt. about 4.5 · 105), which are the smallest biologically active components that can be obtained. 3. 3. The dissociation is reversible in the pH range 4–11. Reassociation is induced by readjusting the pH to 5–7 or, at alkaline pH values, by the addition of bivalent cations. 4. 4. The pH-stability pattern and the near-independence of the degree of dissociation from the concentration indicate that dissociation is a co-operative process. 5. 5. The association-dissociation behavior does not obey the rules valid for a simple equilibrium; therefore a certain heterogeneity in the bonds between the structural units must exist. 6. 6. Whole molecules are dissociated into halves by hydrostatic pressure. This results in an elevated baseline between the two components in the Schlieren patterns.

Estrogen-induced phosphoprotein synthesis in roosters: Kinetics of induction

Abstract The synthesis of the lipovitellin-phosvitin phosphoprotein complex induced in the rooster liver by estradiol and its removal from the blood were investigated. The complex is detectable in the blood 10 h after the first estradiol dose and its concentration increases linearly from about 25 h on for several days, followed by a decline to the original undetectably low level. The half-life of complex in the blood of estrogenized roosters is 8.4 h and in that of untreated control roosters 5.2 h as could be shown by the degradation of intravenously injected radioactive complex. In the period of detectable concentrations the rate of synthesis is calculated from the accumulation and degradation data. From at least 25 h after estradiol injection, the rate of synthesis increases linearly for several days. This is interpreted as the synthesis of long-lived mRNA(s) at a constant rate. These data agree with the finding that the amount of lipovitellin-synthesizing polysomes per liver increases linearly in the same period. From both observations a constant translation rate of 6–10 amino acids · s−1 per ribosome can be calculated. A “memory effect” is observed after a second hormone dose given some time after the disappearance of the phosphoprotein complex from the blood. Detectable accumulation starts sooner and its increase is more rapid in the initial period. This phenomenon is not due to a shorter lag period, for the start of induced synthesis, measured by incorporation of 32P1 into phosphoprotein complex, is 3–4 h after the first as well as after the second injection. From that moment the rate of synthesis after the first injection increases only gradually and becomes a linear function of time after about 15 h, whereas after a second injection the rate of synthesis increases linearly immediately after the lag period.

Induction of phosvitin synthesis in roosters by estradiol injection

Abstract The synthesis of phosvitin, induced by injection of estradiol into non-laying hens and roosters, was investigated in vivo by phosphoprotein phosphate estimations in blood plasma samples. Beginning 20 h after injection of the hormone, the level of phosvitin in the blood rose rapidly; the peak level was proportional to the dose of hormone. A second dose of estradiol, given when the level of phosphoprotein phosphate in the blood was falling again, induced a renewed increase after only 6 h. This secondary increase was caused by enhanced synthesis as it was inhibited by cycloheximide. The first part of the secondary increase was not inhibited by actinomycin D: this indicates reactivated translation of phosvitin mRNA still present. Experiments with tritiated serine demonstrated that synthesis and degradation of phosvitin may occur simultaneously.

Structure and properties of hemocyanins. II. Electron micrographs of the hemocyanins of Sepia officinalis, Octopus vulgaris and Cancer pagurus.

Electron micrographs of the hemocyanins of Sepia officinalis, Octopus vulgaris and Cancer pagurus have been taken using the negative-staining technique. The results indicate that the molecules of Sepia and Octopus hemoeyanin at pH 7·8 are cylinders built of three layers perpendicular to a fivefold axis, about 140 A high and 300 A in diameter. They closely resemble half-molecules of Helix pomatia hemoeyanin. Cancer hemoeyanin, which is much smaller, is visualized as a pentalateral prism with ten subunits situated at the corners, resembling somewhat the dissociation products of the larger hemocyanins.

Specific inhibition of ribosomal RNA synthesis in vitro by guanosine 3' diphosphate, 5' diphosphate.

CONSIBERABLE evidence has accumulated that guanosine 3′-diphosphate, 5′-diphosphate (ppGpp)1 is involved in the regulation of ribosomal RNA (rRNA) synthesis in Escherichia coli in vivo (see ref. 2). The involvement of ppGpp is most convincingly indicated by results obtained under conditions of amino acid starvation. A strong increase in ppGpp accumulation is always accompanied by a concomitant decrease in stable RNA accumulation3. In vitro, a specific effect of ppGpp has not been found, neither in a purified4, nor in a crude system5. Travers et al.6,7 have proposed that ppGpp specifically affects rRNA synthesis by counteracting the effects of the protein elongation factor EF-Tu. Here, we show that ppGpp can specifically inhibit rRNA synthesis in vitro using purified RNA polymerase with two templates—DNA prepared by phenol extraction, and nucleoids prepared using methods developed by Pettijohn et al.8.

Amino acid and energy requirements of protein synthesis in rat pancreatic tissue in vitro

Abstract Rat pancreas pieces incubated in vitro were found to synthesize net protein as well as specific enzymes, viz. ribonuclease and protease zymogens. Protein synthesis in this system is dependent on the presence of (some) essential amino acids, especially tryptophan, in the incubation medium. Although ribonuclease very probably does not contain tryptophan the presence of this amino acid is required for its synthesis. The synthesis of total protein and the increases of specific proteins are closely linked, and can be correlated with a concomitant increase in oxygen uptake. Chopped pancreas appeared to deteriorate rapidly during incubation, and did not synthesize significant amounts of net protein or enzymes.