M.Yu. Feigina

The structural basis of the functioning of bacteriorhodopsin: an overview.

Mass transport across biological membranes is a pressing problem of modern physicochemical biology, determining to a considerable extent our understanding of the regulatory and bioenergetic processes in the living cell. Latterly, as well as continued interest in the general aspects and theories of transport and in the study of model systems, one may clearly discern a tendency in this field towards detailed investigation of the proteins directly associated with translocation of ions and molecules, independently of whether in the membrane such proteins serve as carriers, channels or receptors. Such a membrane protein, one of the most interesting and extensively explored, undoubtedly is bacteriorhodopsin. Bacteriorhodopsin, the only protein of the purple membrane of halophilic bacteria which thrive under extremal conditions and effectively utilize light energy in the performance of their vital activity [ 11, has been found to function as a light-driven proton pump, establishing a considerable pH gradient in the membrane, utilized in the synthesis of ATP [2]. Despite available source and relatively small size, bacteriorhodopsin has long foiled attempts to elucidate its structure by numerous protein chemists and physicists, and only very recently has a way been found to decode its amino acid sequence and elucidate the chemical nature of its functionally important groups [3,17]. These recent studies have also given the first indications of how the molecule is packed in the membrane. The present paper is a summing up of our studies, begun in 1976, that have resulted in the complete

The complete amino acid sequence of cytoplasmic aspartate aminotransferase from pig heart

Aspartate aminotransferase (L-aspartate: 2-0x0glutarate aminotransferase, EC 2.6.1.1) is one of the principal pyridoxal-P-containing enzymes that catalyse the transamination reactions [3] representing key steps at the intersection between the metabolic pathways of amino acids and dicarboxylic acids. Although the catalytic mechanism of aspartate aminotransferase has been investigated at the level of substrate-coenzyme models [4], its elucidation in detail requires knowledge of the enzyme’s structure, considering, in particular, that the very high rates of the enzymic process are determined by the structural peculiarities of the specific protein(apoenzyme) of the aspartate aminotransferase. Accordingly, we embarked on the task of elucidating the amino acid sequence of this enzyme. In the present paper the concluding stage of the work is reported*. The object chosen for study was the aspartate aminotransferase of the cytosol of pig heart; the enzyme, which is different from the mitochondrial isozyme [5,6] was prepared by a previously reported procedure [7]. The enzyme is a complex dimeric protein of high molecular weight; each of the associated subunits

PRODUCTS OF LIMITED PROTEOLYSIS OF BACTERIORHODOPSIN GENERATE A MEMBRANE POTENTIAL

In recent years, one of the most interesting events in biochemistry was the discovery of a new type of photosynthesis mediated by bacteriorhodopsin, a light-dependent proton pump in the membrane of halophilic bacteria. The goal of the present work was to elucidate whether the water-exposed sites of the polypeptide chain of bacteriorhodopsin are involved in the generation of a transmembrane electric potential difference. We have studied the products of partial proteolysis of bacteriorhodopsin by papain and thermolysin. The experiments revealed that the products of hydrolysis of bacteriorhodopsin that retain the AsTO,, max are still competent in generating a transmembrane electric potential difference if illuminated with continuous light or with a flash inducing a single bacteriorhodopsin turnover. The kinetic parameters of bacteriorhodopsin as a photoelectric generator do not deteriorate in spite of removal of 17 amino acids from the C-end, 3 amino acids from the N-end and 5 amino acids from the middle part of the polypeptide chain.

Mass spectrometric determination of the amino acid sequence in arginine-containing peptides

Покaзaно, что aргинин-содержaщие пептиды перед их мaсс-спектрометри ровaнием следует преврaщaть в соответсвующие орнитиновые или пиримидилорнити-н овые пептиды путем гидрaзинолизa или гетероциклизaции сβ-дикaрбонильными соединениями.

Mass spectrometric amino acid sequence determination in arginine-containing peptides☆

Abstract Methods of converting arginine residues into ornithine, N δ -pyrimidylornithine, N δ -imidazolidinonidenornithine residues in peptides have been developed. The modified peptides undergo the amino acid type of fragmentation, so that mass spectrometry can now be used for amino acid sequence determination in arginine-containing peptides.

Cytoplasmic aspartate aminotransferase from pig heart muscle: Partial sequence

In an earlier paper [ 1 ] we reported sequence studies on a number of peptides from the tryptic digest of cytoplasmic aspartate aminotransferase (L-aspartate:2_ketoglutarate aminotransferase, EC 2.6.1.1) from pig heart muscle. Amino acid sequences of a further series of tryptic peptides have been determined, some partial sequences have been completed and data are given on isolation and sequence determination of the products of chymotryptic digestion and cyanogen bromide cleavage of CM-AAT.

The rational use of mass spectrometry for amino acid sequence determination in peptides and extension of the possibilities of the method

It has been shown earlier by us and by others that mass spectrometry can be used for the elucidation of the primary structure of peptides containing residues of all the ammo acids commonly found in proteins, N-acylpeptide esters being most suitable for this purpose (see [ 1,2] and references therein). Mass spectrometric determination of amino acid sequences is based on fragmentation of N-acylpeptide esters involving rupture of amide (ester) bonds and localization of the positive charge at the C-terminus of the amino acid fragments of type (I). These fragments decompose further., consecutively eliminating amino acid residues either in one (I + III) or in two steps (I+II+III), in the latter case via aldimine fragments (II). This pattern of fragmentation provides ammo acid and/or aldimine fragment ions arising from the partial cleavage of every amide bond, the difference in m/e of the fragments allowing the determination of the amino acid sequence in the peptide being investigated. The peaks due to

The primary structure of cytoplasmatic aspartate aminotransferase from pig heart muscle tryptic hydrolysis products

Aspartate aminotransferase (Laspartate: 2-ketoglutarate aminotransferase, EC 2.6.1 .l .)*‘plays an important part in the metabolic activity of the cell, acting as a link between carbohydrate and amino acid metabolism. The mechanism of interaction of the AAT coenzyme (pyridoxal S-phosphate) with substrates has been fairly well investigated [ 1, 21, whereas the role of the apoenzyme in the enzymatic activity is still rather obscure partly because the primary structure of AAT is not yet known. To fill the gap we have investigated the total primary structure of cytoplasmatic AAT from pig heart muscle. In this paper we report the results of our work on isolation and determination of partial and total sequences of tryptic peptides constituting the greater part of the polypeptide chain of the AAT subunit. To accomplish this we made use of exhaustive tryptic hydrolysis, cleaving the peptide chain at lysine and arginine residues and restricted tryptic hydrolysis affecting only arginine residues, the lysine residues being blocked by treatment with maleic anhydride [3]. It is known that the AAT subunit is a polypeptide

Complete primary structure of cytoplasmatic aspartate-amino transferase from hog heart muscle

The complete amino acid sequence of the cytoplasmatic aspartate-amino transferase from hog heart muscle was established. The enzyme consists of two identical subunits, each of which represents one polypeptide chain that contains 412 amino acid residues. The molecular weight of one aspartate-amino transferase subunit is 46, 344.

Investigations in the field of depsipeptides Communication 44. Mass spectrometric study of cyclodidepsipeptides-2,5-diketomorpholines

The fragmentation of cyclodidepsipeptides during electron impact was investigated. The nature of the hydroxy and amino acid residues contained in the 2,5-diketomorpholines can be established on the basis of the mass spectra.