Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Lipids as Anchors01:32

Lipids as Anchors

In the plasma membrane, the lipids forming the bilayer can also act as an anchor to tether proteins to the membrane. The three main types of lipid anchors found in eukaryotes are – prenyl groups, fatty acyl groups, and glycosylphosphatidylinositol or GPI groups. Prenyl and fatty acyl groups act as anchors on the cytosolic surface of the membrane, whereas GPI anchors proteins on the extracellular side.
The carboxy-terminal of most of the prenylated proteins, such as Ras proteins, contains the...
ER Retrieval Pathway01:45

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms...
Conserved Binding Sites01:49

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Conserved Binding Sites01:49

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
Cytoskeletal Accessory Proteins01:13

Cytoskeletal Accessory Proteins

The cytoskeleton is an essential cell component that plays several structural and functional roles. However, the filaments that make up the cytoskeleton cannot function independently and depend on the accessory or ancillary proteins to effectively carry out their function. Accessory proteins associate with cytoskeletal filaments and their monomers, aiding filament formation and function. They also help in the cross-communication among cytoskeletal filaments. Cytoskeletal accessory proteins are...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

MORC3 represses a tandem repeat enhancer to regulate interferon.

The EMBO journal·2026
Same author

MiR-182-5p, miR-122-5p and hsa-piR-28004 as indicators of disease and treatment in pediatric familial hypercholesterolemia.

Pediatric research·2026
Same author

In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.

The Journal of experimental medicine·2026
Same author

Retinol Binding Protein 4 reactivates latent HIV-1 by triggering canonical NF-κB, JAK/STAT5 and JNK signalling.

Signal transduction and targeted therapy·2025
Same author

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes.

Nature communications·2025
Same author

A cereblon-based glue degrader of NEK7 regulates NLRP3 inflammasome in a context-dependent manner.

Cell chemical biology·2025

Related Experiment Video

Updated: Jul 3, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

A conserved cysteine motif essential for ceramide kinase function.

Emilie Lidome1, Christine Graf, Markus Jaritz

  • 1Novartis Institutes for BioMedical Research, Vienna, Vienna, Austria.

Biochimie
|July 30, 2008
PubMed
Summary

Ceramide kinase (CerK), an enzyme sensitive to oxidation, has its key functional cysteines identified. A specific cysteine cluster (C347XXXC351XXC354) is crucial for CerK activity.

More Related Videos

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability
12:26

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Published on: June 2, 2023

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
11:25

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Published on: October 4, 2017

Related Experiment Videos

Last Updated: Jul 3, 2026

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability
12:26

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Published on: June 2, 2023

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
11:25

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Published on: October 4, 2017

Area of Science:

  • Biochemistry
  • Enzymology
  • Sphingolipid Metabolism

Background:

  • Ceramide kinase (CerK) is a vital enzyme in sphingolipid metabolism.
  • CerK is known to be highly sensitive to oxidative damage, but the specific residues involved are unidentified.

Purpose of the Study:

  • To identify the specific cysteine residues in CerK that are critical for its enzymatic activity and sensitive to oxidation.
  • To elucidate the structural determinants of CerK's oxidative sensitivity.

Main Methods:

  • Utilized N-ethyl-maleimide, a thiol-modifying agent, to assess CerK activity in vitro and in cell-based assays.
  • Performed site-directed mutagenesis, replacing residues with alanine, to generate CerK mutants.
  • Assessed the enzymatic activity of CerK mutants.

Main Results:

  • N-ethyl-maleimide treatment abolished CerK activity, indicating accessible cysteine residues.
  • A critical cluster of cysteine residues, C(347)XXXC(351)XXC(354), was identified as essential for CerK function.
  • Homology modeling provided insights into the catalytic domain structure concerning these cysteines.

Conclusions:

  • The identified cysteine cluster is indispensable for ceramide kinase activity.
  • These findings reveal key structural features contributing to CerK's sensitivity to oxidation.
  • Understanding these determinants can inform strategies to protect CerK function.