Søren G. Laland

On the presence of two new high mobility group-like proteins in HeLa S3 cells

Two phosphorylated HMG‐like proteins with M r ≈ 10 000 have been isolated from HeLa S3 cells, one being present in metaphase and one in interphase cells. The amino acid compositions of these proteins are very similar but differ from the known HMG proteins. However, they exhibit similarities being rich in proline, basic and acidic amino acids. A possible role in chromatin condensation of the HMG‐like protein characteristic for metaphase cells is suggested.

The presence and possible role of phosphopantothenic acid in gramicidin S synthetase

Orn + Leu [5]. In agreement with this finding it was shown independently by Gevers, Kleinkauf and Lipmann [4] and FrQyshov, Zimmer and Laland [3] that di-, tri-, tetra-, and pentapeptides all having D-phenylalanine at the N-terminal end aswell as all five amino acids in gramicidin S are bound to the enzyme through thio- ester linkages. The possible presence of 4’-phosphopantetheine in gramicidin S synthetase occurred to us some time ago when evidence for the presence of acid stable and alkali labile enzyme intermediates was obtained [ 1, 21. Preliminary analysis of a gramicidin S synthetase preparation, designated fraction 5 and *

On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells.

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.

The nature of the enzyme bound intermediates in gramicidin s biosynthesis

Previous work [ 1,2] indicated that during synthesis, intermediates are covalently bound to grarnicidin S synthetase. Furthermore, the synthesis starts with phenylalanine and the sequence of addition of amino acids is Phe-Pro-Val-Orn [2]. When the present work was completed, a paper by Gevers, Kleinkauf and Lipmann [3] appeared which indicated that all fne amino acids and the intermediate peptides D-Phe-L-Pro, D-Phe-L-Pro-L-Val, D-Phe-LPro-L-Val-L-Om, and D-Phe-L-Pro-L-Val-LOrn-L-Leu are bound covalently to the synthetase, most probably through thioester linkages. This work presents additional evidence for this view. The present results also show that there is a marked difference in the stability of the linkages of ornithine and D-PheL-Pro to the protein towards ethanolHC1 compared to that of the other covalently bound intermediates. Using a different method to that of Gevers et al. [3] no evidence for peptides longer than the pentapeptide was found. This finding supports the view that gramicidin S is formed by head to tail condensation of activated pentapeptides

The human chromosomal protein HMG I contains two identical palindrome amino acid sequences

The sequence of 105 amino acids of the human high mobility group chromosomal protein HMG I has been determined. The most striking feature of this sequence is two identical palindrome sequences: pro-arg-gly-arg-pro, which together with a third related sequence: gly-arg-pro-arg, may represent the binding sites of HMG I to clusters of A-T base pairs in DNA.

Phosphorylation of P1, a high mobility group‐like protein, catalyzed by casein kinase II, protein kinase C, cyclic AMP‐dependent protein kinase and calcium/calmodulin‐dependent protein kinase II

P1, a high mobility group‐like nuclear protein, phosphorylated by casein kinase II on multiple sites in situ, has been found to be phosphorylated in vitro by protein kinase C, cyclic AMP‐dependent protein kinase and calcium/calmodulin‐dependent protein kinase II on multiple and mostly distinct thennolytic peptides. All these enzymes phosphorylated predominantly serine residues, with casein kinase II and protein kinase C also labeling threonine residues. Both casein kinase II and second messenger‐regulated protein kinases, particularly protein kinase C, might therefore be involved in the physiological regulation of multisite phosphorylation of PI.

[43] Gramicidin S synthetase

Publisher Summary This chapter discusses the assay and purification procedure of gramicidin S synthetases. The following enzymic activities may be used for assaying gramicidin S synthetase: (1) synthesis of gramicidin S; (2) amino acid-dependent ATP- 32 PPi exchange; (3) amino acid-dependent ATP-[ 14 C] AMP exchange; (4) thioesterbonding of the individual amino acids, (5) ATP-dependent racemization of phenylalanine. Purification procedure involves cultivation of the microorganism, preparation of crude extract, streptomycin sulfate precipitation, ammonium sulfate precipitation, and chromatography on DEAE sephadex A-50, chromatography on sephadex G-200 alternative method or the separation of light and heavy enzyme: affinity chromatography. The biosynthesis of gramicidin S is the result of the concomitant functioning of a large number of catalytic activities. For instance, in the case of the heavy enzyme it has been estimated that about 18–20 different catalytic activities are involved. During the purification of gramicidin S synthetases that gives an almost homogeneous preparation of heavy enzyme, some 97% of its ability to synthesize the antibiotic is lost. The loss is particularly great during fractionation on the DEAE Sephadex G-50 and Sephadex G-200. Of the many catalytic activities involved in the biosynthesis, the catalytic activities responsible for amino acid activation are remarkably stable.

Isolation of a peptide conjugate with the sequence Phe-Pro-Val-Orn from a cell-free system producing gramicidin S

Abstract 1. 1. A peptide conjugate with the suggested structure Phe-Pro-Val-Orn-R has been isolated from a cell-free system of Bacillus brevis synthesizing gramicidin S. The nature of R is unknown. Available evidence suggests that it is linked to the carboxyl group of ornithine. 2. 2. In a cell-free system, which synthesizes gramicidin S, the phenylalanine analogue, thienylserine, reduces the incorporation of [14C]valine into gramicidin much more effectively than [14C]leucine. One possible explanation is i.e. the existence, in the cell-free system, of a tetrapeptide with the sequence Phe-Pro-Val-Orn.

The metaphase specific phosphorylation of HMG I

In vivo labelling of HeLa cells arrested in metaphase with [32P]-phosphate and in vitro phosphorylation of HMG I with the partially purified growth associated H1 kinase was used to study metaphase specific phosphorylation of HMG I. It was found that threonine 53 and 78 became phosphorylated. These amino acids are embedded in respectively the sequence PTPKR and TPGRK which are similar to the sequences phosphorylated by the growth associated H1 kinase.

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs.

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.