C. M. Hoogerbrugge

Characterization of O‐Glycosylated Precursors of Insulin‐like Growth Factor II by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry

High molecular weight precursors of insulin-like growth factor II (IGF-II) were isolated from Cohn fraction IV of human plasma by ultrafiltration, affinity chromatography and reversed-phase high-performance liquid chromatography. Molecular weight determination by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of two high molecular weight IGF-II preparations revealed heterogeneous glycosylation. A combination of enzymatic degradation and MALDI-MS were applied for further structural characterization of the glycosylated precursors of IGF-II. The first step was molecular weight determination of intact high molecular weight IGF-IIs prior to and after treatment with neuraminidase and O-glycosidase. This, together with a comparison of molecular weight information available from the cDNA, revealed that both high molecular weight IGF-II species contain an identical C-terminal extension of 20 residues but different degrees of glycosylation. Second, comparative Endo Glu-C digestion of the preparations prior to and after enzymatic release of carbohydrates and subsequent remeasurement of the molecular weight by MALDI-MS confirmed the primary structure of precursor IGF-II1-87. The O-linked carbohydrates were found to be associated with the C-terminal extension and the heterogeneity was identified as varied sialylated forms of one and two HexNAc-Hex groups.

Molecular aspects of the human somatomedins.

The primary structure of the somatomedins (SM) IGF-I and IGF-II has been known for some years. Both from isolation of the SMs from plasma and from the study of cDNAs it has become evident that there are more SMs, needing further study. In plasma the SMs are present in larger molecules of two size-classes: 150 kD and 40 kD, behaving as specific SM-binding proteins. The possibility that such protein classes are heterogeneous and also contain precursor molecules undergoing proteolytic processing is considered. The somatomedin genes are discontinuous and span large regions of the human genome.

Co-administration of IGF-binding protein-3 differentially inhibits the IGF-I-induced total body and organ growth of Snell dwarf mice

In mammals IGF-I is part of a 150-kDa binding protein complex, which also contains a glycosylated acid-labile protein (ALS) and a glycosylated acid-stable IGF binding subunit IGFBP-3. Administration of free IGF-I in vivo induces not only acute insulin-like effects but also growth stimulation. Since co-injection with IGFBP-3 only partially blocked the hypoglycemic response of free IGF-I in hypophysectomized rats, we were interested in the growth stimulating activity of the IGFI-IGFBP-3 complex in pituitary-deficient mice compared to that obtained by IGF-I alone. Therefore, the effects of subcutaneously administered IGF-I, IGFBP-3 and the IGF-I-IGFBP-3 complex on somatic growth and organ growth of pituitary-deficient Snell dwarf mice were studied after 4 weeks of treatment. Treatment with IGF-I alone induced a significant increase in body length and weight, as well as in weights of the submandibular salivary glands, kidneys and quadriceps femoris muscles as compared to buffer treated controls. No significant changes were found in liver, brain, heart and thymus. IGFBP-3 alone had no effect. However, the stimulating effects of IGF-I alone on body length and weight, as well as on the weight of the kidneys, were fully neutralized by co-injection with IGFBP-3. In contrast, the weights of submandibular salivary glands and m. quadriceps femoris were increased by treatment with the complex compared to controls and not significantly different from animals treated with IGF-I alone. Our data show that in GH-deficient mice administration of IGFBP-3 differentially inhibits the IGF-I induced body and organ growth. This calls for extra vigilance when exploring presumed advantages of administering an IGF-I-IGFBP-3 complex to GH-deficient individuals in order to obtain stimulation of growth.

Evidence for carrier ampholyte-peptide interactions during somatomedin purification

Abstract The application of gel filtration on Sephadex G-50, hydrophobic interaction on octyl-Sepharose and mixed ion-exchange on AG 50 1-X8 wre examined for the removal of carrier ampholytes from peptide mixtures containing somatomedins. With respect to gel filtration, the results suggest peptide—ampholyte interactions, particularly at pH 2.5, which prevent complete separation. Hydrophobic interaction chromatography and mixed ion-exchange chromatography appear potentially suitable, although the use of th latter is limited to peptides having molecular weights > 4000 daltons.

The effect of hGH, thyroxine and semipurified somatomedin (SM) on cellular growth of some organs of the Snell dwarf mouse

Growth of the hypopituitary Snell mouse is partially restored by administration of hGH, thyroxine and SM. In order to investigate whether these preparations induce cell division and/or hypertrophy DNA and protein of liver,kidney and spleen were measured after treatment and contrasted with normal and dwarfed controls. Results of the latter are as follows:SM induces cell division but only little cell enlargement in the liver and the spleen, as do hGH and T4. Cell division of the kidney is barely affected by SM and hGH, but markedly by T4, whereas all preparations induce hypertrophy of this organ.

Primary sequences of insulin-like growth factors 1 and 2 isolated from porcine plasma

Insulin-like growth factors 1 and 2 were purified from porcine plasma. In addition to the determination of their isoelectric points, the primary structures of both proteins were determined, using low microgram quantities of protein, by the versatile combination of time-of-flight plasma desorption mass spectrometry and automated Edman degradation. Porcine insulin-like growth factor 1 was shown to be homologous to both human and bovine proteins; the type 2 growth factor showed one mutation to both human and bovine type 2 proteins.

OBSERVATIONS ON A SOMATOMEDIN (SM) INHIBITOR IN SEVERELY MALNOURISHED CHILDREN

In 13 patients with marasmus or kwashiorkor, plasma SM was measured using the porcine costal cartilage assay. Non-parallelism (N.P.) due to a shallow slope prevented quantitation in many samples. Results: SM-distriSution: on admission ≤ .20:5; .20 < x < .50:3; .50 < x < .80:2, N.P.:3. After 4-6 weeks of treatment: low : 6, normal: 1, N.P. : 3 (3 had died, all with initial SM < .20). After 8-10 weeks: low: 3, normal or high : 3, N.P. : 3 (one more child had died). Mixing plasma from 3 untreated patients with the standard lowered its apparent potency with 35, 67 and 0% resp. Heating plasma after acidification increased its potency relative to a standard control from < .12 to .65. Concl.: In severe and chronic malnutrition heat-labile SM-inhibiting material is present in plasma, initially persisting during treatment. This may contribute to the problems in reverting these children from a katabolic to an anabolic state.