Jean Petre

Purification and properties of an endo-β-1,4-glucanase from Clostridium thermocellum

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate which, until now, has not been resolved into individual enzyme components. By using QAE-Sephadex A50 chromatography in the presence of 6 M urea, it was possible to split the complex into distinct protein fractions. One of these fractions contained an endo-beta-1,4-glucanase which was isolated at a high degree of purity and was identified by its ability to hydrolyze trinitrophenylated carboxymethylcellulose. The enzyme is of monomeric nature, with a molecular weight of 56,000. It has an isoelectric pH of 6.2 and an optimum pH of 6.0. It hydrolyzed carboxymethylcellulose and, at a slower rate, cellulose powder. The major end products of cellulose degradation are glucose, cellobiose and cellotriose; cellotetrose is formed as an intermediate product. No specific small molecular weight activator or inhibitor was found except cellobiose and, to a lesser extent, glucose, which at high concentrations partially inhibit the activity of the enzyme. The temperature dependence of the enzyme is related to the thermophilic character of the producing microorganism.

Complex formation between transfer RNAs with complementary anticodons: Use of matrix bound tRNA

Abstract It is shown that yeast tRNAPhe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNAPhe in its ability to form a specific complex with E. coli tRNA2Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpAOH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli

SummaryThe conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).

Protein-chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin-binding subunit

The purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S1, free S5 and the free complexes S2‐S4 and S3‐S4, with S2‐S4 as the only haptoglobin‐binding moiety, identifying S2 as the haptoglobin‐binding protein. Partial NH2‐terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS‐polyacrylamide gel electrophoresis followed by electroblotting onto polybrene‐coated glass‐fiber sheets. The sequences reveal extensive homology of the N‐terminal portions of the constitutive subunits S2 and S3 and between S1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli.

Direct biochemical approach to the structural heterogeneity of 30 S ribosomes from Escherichia coli.

Structural heterogeneity of purified 30 S ribosomes was demonstrated by Voynow and Kurland [l] several years ago. Stoichiometry measurements indicated that three main classes of proteins exist in the 30 S particle: the fractional ones, which are present in amounts corresponding to 0.5 or fewer copies per ribosome; the unit proteins, which are present in amounts equal or very close to one copy per ribosome and the marginal ones, with intermediate stoichiometry or for which precise data are not available. A definite decision on whether the 30 S ribosome is also heterogeneous in viva cannot be made in the present state of knowledge, neither is it definitely proved that the structural heterogeneity found in vitro is not an artifact due to the experimental procedures. One of the most attractive hypotheses to interpret the structural heterogeneity supposes that the 30 S subunit cycles through different states during protein synthesis, the idea being that there is only one class of 30 S ribosomes which undergo structural changes during the initiation, elongation and termination processes [2,3]. An alternative explanation suggests, on the contrary, that the composition of 30 S ribosomes does not change during protein synthesis; in this case. one may imagine that the fractional proteins either are unimportant ones, or serve to differentiate ribosomes into different classes with different specificities or regulatory properties.

The binding of Escherichia coli ribosomes to matrix bound messenger RNA

Summary The formation of a hydrazone derivative between periodate oxidized RNA and a linear polymer of acrylic hydrazide entrapped in an agar gel affords a convenient method to prepare a resin bearing chemically coupled synthetic messenger RNA. The use of such an RNA derivative instead of the free messenger makes it possible to isolate 30S or 70S ribosomes involved in the initiation complex programmed by poly(A,G,U) as messenger, and of 70S ribosomes bound to a poly(U) template. The complex isolated is specific and, as judged by the puromycin reaction, it retains a high activity to transfer bound N-blocked aminoacyl-transfer RNA. Therefore, this method appears an unique way to prepare a population of active ribosomes presumably homogeneous with respect to their functional state.