Ella Cederlund

Analysis of an inhibin preparation reveals apparent identity between a peptide with inhibin-like activity and a sperm-coating antigen

The amino acid sequence of a large form of inhibin‐like peptide in human seminal plasma was determined, and compared with structures reported for similar inhibin preparations and a seminal plasma globulin. The data confirm and correlate previous reports on this form of inhibin‐like peptide. The structural comparisons further suggest that the peptide is closely similar to or possibly identical to a sperm‐coating antigen reported to be synthesized from prostatic epithelium. This may correlate with non‐gonadal origins of inhibin‐like material and will help to elucidate the biological roles of inhibin(s).

Class IV mammalian alcohol dehydrogenase: Structural data of the rat stomach enzyme reveal a new class well separated from those already characterized

The stomach form of alcohol dehydrogenase has been structurally evaluated by peptide analysis covering six separate regions of the rat enzyme. Overall, this new structure diners widely (32–40% residue differences) from the structures of three classes of alcohol dehydrogenase characterized before from the same species. Consequently, this novel enzyme constitutes a true fourth class of mammalian alcohol dehydrogenase. In particular, differences are extensive also towards class II, although enzymatic and physicochemical properties initially suggested overall similarities with class II. The new structure establishes the presence of one further alcohol dehydrogenase mammalian gene, extends the enzyme family derived from repeated gene duplications, and confirms tissue‐specific expressions.

Class IV alcohol dehydrogenase (the gastric enzyme) : structural analysis of human σσ-ADH reveals class IV to be variable and confirms the presence of a fifth mammalian alcohol dehydrogenase class

Human gastric alcohol dehydrogenase (σσ‐ADH) was submitted to peptide analysis at picomole scale. A total of 72 positions were determined in the protein chain, providing information on three aspects of alcohol dehydrogenase structures in general. First, the data establish the presence of a unique class of the enzyme, now confirmed as class IV, expressed in gastric tissue and separate from another novel class. now termed class V. Second, the class IV gastric enzyme has active site relationships compatible with an ethanol‐active, zinc‐containing alcohol dehydrogenase. Third, this enzyme class is of the variable type, like that for the ‘variable’, classical liver alcohol dehydrogenase of class I, and in contrast to that for the ‘constant’ class III enzyme. Known human alcohol dehydrogenase structures now prove the presence of at least seven human genes for the enzyme and nine for the whole protein family.

Peptides of postulated inhibin activity: Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment

A 94‐residue polypeptide isolated from human seminal plasma and its chemically synthesized C‐terminal 28‐residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH‐stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94‐residue peptide and the synthetic replicate of its C‐terminal 28‐residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.

Structural and Enzymatic Properties of a Gastric NADP(H)- dependent and Retinal-active Alcohol Dehydrogenase*

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP+ rather than NAD+. Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP+ preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k cat/K m = 3.4·104 mm −1min−1). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58–60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Characterization of carrot pectin methylesterase.

Abstract. The most alkaline form of pectin methylesterase was purified from ripe carrot roots and used for structural analysis. Determination of an N-terminal blocking group and of the primary structure allowed comparisons with other forms, and facilitated crystallographic determination of the three-dimensional structure. The mature enzyme has 319 residues and the N-terminal blocking group was shown to be a pyroglutamyl residue derived from a glutaminyl cyclization. Few other methylesterases have been isolated and assigned to exact mature forms, and together with the present enzyme, only two have been analyzed in three-dimensional structure. However, comparison of 39 forms, mainly from GenBank data, reveals clear relationships and identifies sub-groups of this enzyme type, deviating in structure but centering around two functionally important and conserved Asp residues at positions 136 and 157 in the carrot enzyme.

Activity of the plant peptide aglycin in mammalian systems

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27–63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu‐C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 µg·g−1 body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage‐dependent anion‐selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.

CHARACTERIZATION OF HUMAN DEOXYCYTIDINE KINASE : CORRELATION WITH CDNA SEQUENCES

Existing data on the structure of human deoxycytidine kinase (dCK) diverge. A monomeric 60 kDa form has been isolated and the cloning of a cDNA coding for 626 amino acids corresponding to a 71 kDa protein has been reported. However, pure dCK isolated from leukemic spleen is a dimer of 30 kDa subunits. Amino acid sequences of peptides from digests of this protein are now presented. None of the peptide structures obtained correspond to the cDNA for the 71 kDa protein, but to a cDNA for a 30.5 kDa dCK recently cloned. Furthermore, homology of the peptide sequences of dCK to parts of thymidine kinases and protein‐tyrosine kinases are detected.

11-Hydroxythromboxane B2 dehydrogenase is identical to cytosolic aldehyde dehydrogenase

11 ‐Hydroxythromboxane B2 dehydrogenase purified from porcine kidney has been identified as cytosolic aldehyde dehydrogenase (EC 1.2.1.3). This identification is based on protein characteristics, sequence analysis of one proteolytic digest, blocked N‐terminus, subunit molecular mass of 55 kDa, and enzymatic activities. The sequence difference with the human enzyme is 7.5% in the fragments analyzed (29 exchanges of 388 positions, corresponding to the expected species variability for cytosolic aldehyde dehydrogenase The substrate thromboxane B2 contains a hemiacetal in its ring structure, but the reaction most likely proceeds via the aldehyde form of the substrate. This finding is in agreement with the proposed metabolism of 4‐hydroxycyclophosphamide and highlights the possibility that molecules containing a hemiacetal structure can function as substrates for aldehyde dehydrogenase.